Zika vaccines and immunogenic compositions, and methods of using the same

ABSTRACT

The present disclosure relates to Zika virus vaccines and immunogenic compositions having one or more antigens from a Zika virus (e.g., a Zika virus clonal isolate, a non-human cell adapted Zika virus, etc.), and methods of treatments and uses thereof

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under Contract No. HHSO100201600015C with the Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority. This invention was created in the performance of a Cooperative Research and Development Agreement with the Centers for Disease Control and Prevention, an Agency of the Department of Health and Human Services. The Government of the United States has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to Zika virus vaccines and immunogenic compositions having one or more antigens from a Zika virus (e.g., a Zika virus clonal isolate, a non-human cell adapted Zika virus, etc.) and methods of treatment and uses thereof.

BACKGROUND

Zika virus, a flavivirus classified with other mosquito-borne viruses (e.g., yellow fever, dengue, West Nile, and Japanese encephalitis viruses) within the Flaviviridae family has spread rapidly in a hemispheric-wide epidemic since the virus was introduced into Brazil in 2013. The virus has reached the Central and North Americas, including territories of the United States, consequently now threatening the continental US. Indeed, Zika virus strain PRVABC59 was isolated from serum from a person who had traveled to Puerto Rico in 2015. The genome of this strain has been sequenced at least three times (See Lanciotti et al. Emerg. Infect. Dis. 2016 May; 22(5):933-5 and GenBank Accession Number KU501215.1; GenBank Accession Number KX087101.3; and Yun et al. Genome Announc. 2016 Aug. 18; 4(4) and GenBank Accession Number ANK57897.1).

Initially isolated in 1947 in Uganda, the virus was first linked to human disease in 1952, and has been recognized sporadically as a cause of mild, self-limited febrile illness in Africa and Southeast Asia (Weaver et al. (2016) Antiviral Res. 130:69-80; Faria et al. (2016) Science. 352(6283):345-349). However, in 2007, an outbreak appeared in the North Pacific island of Yap, and then disseminated from island to island across the Pacific, leading to an extensive outbreak in 2013-2014 in French Polynesia, spreading then to New Caledonia, the Cook Islands, and ultimately, to Easter Island. An Asian lineage virus was subsequently transferred to the Western Hemisphere by routes that remain undetermined (Faria et al. (2016) Science. 352(6283):345-349). The virus may be transmitted zoonotically by Aedes aegypti, A. albopictus, and possibly by A. hensilli and A. polynieseinsis (Weaver et al. (2016) Antiviral Res. 130:69-80). Additionally, it is thought that other vectors for transmitting the virus may exist, and the virus may be transmitted by blood transfusion, transplacentally, and/or through sexual transmission.

In late 2015, a significant increase in fetal abnormalities (e.g., microcephaly) and Guillain-Barre syndrome (GB S) in areas of widespread Zika virus infection raised alarm that Zika virus might be much more virulent than originally thought, prompting the World Health Organization (WHO) to declare a Public Health Emergency of International Concern (PHEIC) (Heymann et al. (2016) Lancet 387(10020): 719-21). Although the WHO has since declared an end to the PHEIC, Zika continues to pose in particular a significant threat for pregnant women and their unborn babies.

While Zika virus poses a substantial public health threat, no FDA-approved vaccine or treatment currently exists, and the only preventative measures for controlling Zika virus involve managing mosquito populations.

In recent efforts to characterize a recombinant Zika virus for the development of a potential vaccine, a non-human cell adapted Zika virus was identified that harbors a mutation in the viral Envelope protein at position 330 (Weger-Lucarelli et al. (2017) Journal of Virology 91(1): 1-10). The authors of this study found that full-length infectious cDNA clones of Zika virus strain PRVABC59 were genetically unstable when amplified during cloning, and opted to split the viral genome to address the observed instability, developing and applying a two plasmid system. However, a two plasmid system for the development of a Zika vaccine is less desirable.

BRIEF SUMMARY

Thus, there is a need to develop vaccines and immunogenic compositions for treating and/or preventing Zika virus. Accordingly, certain aspects of the present disclosure relate a vaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus, wherein the antigen is an inactivated whole virus.

Accordingly, certain aspects of the present disclosure relate a vaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus, wherein the Zika virus comprises at least one non-human cell adaptation mutation.

Accordingly, certain aspects of the present disclosure relate a vaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus, the Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1.

To meet the above and other needs, the present disclosure is directed, at least in part, to a genetically stable non-human cell adapted Zika virus harboring an adaptation in the Non-structural protein 1 (with a wild-type Envelope protein), allowing for the use of a single virus/viral genome system for vaccine production. The present disclosure is also directed, at least in part, to a Zika virus clonal isolate that is genetically homogenous and/or has been purified away from one or more adventitious agents. Accordingly, the present disclosure provides vaccines and immunogenic compositions useful for treating and/or preventing Zika virus infection (in humans) that include one or more Zika virus antigens (e.g., one or more antigens from a whole inactivated Zika virus) from a Zika virus harboring at least one non-human cell adaptation mutation (e.g., a mutation in Zika virus Non-structural protein 1) and/or a Zika virus clonal isolate.

The present disclosure is based, at least in part, on the surprising finding that both high and low dose vaccines comprising one or more antigens from separately derived clonal virus populations of non-human cell adapted Zika virus were able to induce robust immune responses and provide significant protection from Zika virus infection (See Examples 2 and Example 6 below). Clonal isolation of the Zika virus strains also allowed for: 1) the successful purification of the virus away from contaminating agents (e.g., adventitious agents that may be co-purified with the parental strain), and 2) the production of a genetically homogeneous viral population. Moreover, the present disclosure is based, at least in part, on the finding that clonal isolated Zika viruses harboring an adaptation mutation in protein NS1 grew well and predictably in Vero cells to high titer, and surprisingly, were genetically stable/genetically homogenous without any detectable mutations in the viral envelope protein (See Examples 1 and 2 below). While a similar mutation in Zika virus Non-structural protein 1 may have been observed in the genomic sequencing analysis of 1 out of 3 published sequences of Zika virus strain PRVABC59 (Yun et al. Genome Announc. 2016 Aug. 18; 4(4)), this reference fails to teach or suggest that a mutation in NS1 may improve stability of the virus; fails to teach or suggest that a virus harboring the mutation may be used in the development of an effective vaccine against Zika virus; and fails to teach or suggest that such a vaccine may be effective in inducing a robust immune response and providing significant protection from Zika virus infection when used at both low and high doses. Thus, without wishing to be bound by theory, the inventors of the present disclosure have determined that the adaptation mutation in protein NS1 appeared to enhance genetic stability within the Zika virus, resulting in increased/enhanced replication efficiency. Further, the Zika strain harboring an adaptation mutation in protein NS1 was able to be passaged multiple times without developing further mutations. Such a stable Zika virus strain is advantageous as a master virus seed (MVS), or subsequent seeds derived from the MVS, for vaccine production and manufacturing, as the risk of the master virus seed developing undesirable mutations is reduced. Moreover, without wishing to be bound by theory, the adaptation mutation in protein NS1 of the Zika strain of the present disclosure may also reduce or otherwise inhibit the occurrence of undesirable mutations, such as a mutation within the envelope protein E (Env) of the Zika virus strain.

Accordingly, certain aspects of the present disclosure relate to a vaccine or immunogenic composition containing one or more antigen from a Zika virus, where the Zika virus contains at least one non-human cell adaptation mutation. In some embodiments, the at least one non-human cell adaptation mutation is in Zika virus Non-structural protein 1 (NS1). In some embodiments, the at least one adaptation mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the at least one adaptation mutation is a Trp98Gly mutation.

Accordingly, certain aspects of the present disclosure relate to a vaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus, wherein the Zika virus comprises at least one non-human cell adaptation mutation.

Accordingly, certain aspects of the present disclosure relate a vaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus, the Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1.

In some embodiments that may be combined with any of the preceding embodiments, the at least one adaptation mutation enhances genetic stability as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments that may be combined with any of the preceding embodiments, the at least one adaptation mutation enhances viral replication as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments that may be combined with any of the preceding embodiments, the Zika virus does not comprise a mutation in Envelope protein E (Env).

In some embodiments that may be combined with any of the preceding embodiments, the non-human cell is a mammalian cell. In some embodiments that may be combined with any of the preceding embodiments, the non-human cell is a monkey cell. In some embodiments, the monkey cell is from a Vero cell line. In some embodiments, the Vero cell line is a WHO Vero 10-87 cell line.

In some embodiments that may be combined with any of the preceding embodiments, the Zika virus is an African lineage virus or an Asian lineage virus. In some embodiments, the Zika virus is an Asian lineage virus. In some embodiments, the Zika virus is from strain PRVABC59.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is an inactivated whole virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika which differs from strain PRVABC59 in a Trp98Gly mutation at position 98 of SEQ ID NO: 1.

In some embodiments that may be combined with any of the preceding embodiments, the virus was chemically inactivated. In some embodiments, the virus was chemically inactivated with one or more of a detergent, formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetyl ethyleneimine, methylene blue, and psoralen. In some embodiments, the virus was chemically inactivated with formalin.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition further contains an adjuvant. In some embodiments, the adjuvant is selected from aluminum salts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA), synthetic lipid A, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and/or Incomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the adjuvant is selected from the group consisting of alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the one or more antigens are adsorbed to the adjuvant.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is a low or medium dose vaccine or immunogenic composition (e.g., containing from about 1 μg to about 5 μg antigen or 2 μg antigen or 5 μg antigen). In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is a high dose vaccine or immunogenic composition (e.g., containing about 10 μg antigen). In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition contains from about 1 μg to about 25 μg of the one or more antigens, in particular 2 μg, 5 μg or 10 μg, or in particular 10 μg of the one or more antigens. In certain such embodiments the antigen is a purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2. In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate. In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition contains from about 0.1 μg to about 100 μg Zika virus antigen or Env. In some embodiments, the vaccine or immunogenic composition is unadjuvanted. In some embodiments that may be combined with any of the preceding embodiments, the Zika virus is a clonal isolate. In some embodiments, the clonal isolate is substantially free of one or more adventitious agents (e.g., free of one or more adventitious agents that may be co-purified with the parental strain).

Other aspects of the present disclosure relate to a vaccine comprising a Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the vaccine comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine comprises a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2. In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate.

Other aspects of the present disclosure relate to a vaccine or immunogenic composition containing: a) an aluminum salt adjuvant; and b) a purified inactivated whole Zika virus, where the Zika virus contains a non-human cell adaptation mutation, and where the non-human cell adaptation mutation is a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1.

Other aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject in need thereof, including administering to the human subject a therapeutically effective amount of any of the vaccines or immunogenic compositions described herein.

Other aspects of the present disclosure relate to a method for inducing an immune response in a human subject in need thereof, including administering to the human subject an immunogenic amount of any of the vaccines or immunogenic compositions described herein.

In one aspect the present disclosure relates to a method of treating or preventing, in particular preventing Zika virus infection in a human subject in need thereof, comprising administering to the human subject the vaccine or immunogenic composition.

In one aspect the present disclosure relates to a method for inducing an immune response against a Zika virus antigen in a human subject in need thereof, comprising administering to the human subject the vaccine or immunogenic composition.

In one aspect the present disclosure relates to a method of preventing Zika virus disease in a human subject in need thereof, comprising administering to the human subject the vaccine or immunogenic composition. In this case the disease relates to mild fever, maculopapular rash, conjunctivitis and arthralgia. Furthermore, the Zika virus is a neurotropic flavivirus that can potentially cause disease within the central nervous system and Guillain-Barre Syndrome (GBS).

In one aspect the present disclosure relates to a method of preventing Zika virus disease in a fetus or newborn in need thereof, comprising administering to the pregnant human subject or a human subject that intends to become pregnant or woman of childbearing potential the vaccine or immunogenic composition. The Zika disease in this case relates to serious outcomes for the fetus and newborn. The spectrum of congenital anomalies associated with Zika virus infection, known as Congenital Zika Syndrome (CZS), consists of severe microcephaly with partially collapsed skull, cerebral cortices with subcortical calcifications, macular scarring and focal pigmentary retinal mottling, congenital contractures, and marked early hypertonia with symptoms of extrapyramidal involvement.

In one aspect the present disclosure relates to a vaccine or immunogenic composition of for use in a method of treating or preventing, in particular preventing Zika virus infection in a human subject in need thereof, in a method for inducing an immune response against a Zika virus antigen in a human subject in need thereof, and in a method of preventing Zika virus disease in a human subject, fetus or newborn in need thereof.

In one aspect the present disclosure relates to the use of the vaccine or immunogenic composition in the manufacture of a medicament for a method of treating or preventing, in particular preventing Zika virus infection in a human subject in need thereof, a method for inducing an immune response in a human subject in need thereof, and for a method of preventing Zika virus disease in a human subject fetus or newborn in need thereof.

Within the meaning of this disclosure, the treatment includes the treatment of a single human subject and the treatment of a human subject population. A human subject population is considered to encompass the treatment of more than one individual, e.g. 2 or more individuals.

Accordingly, certain aspects of the present disclosure relates to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising an antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the boost administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or of at least 20 Zika virus seronegative human subjects of greater than 300, or greater than 500, or greater than 1000, or greater than 1500, or greater than 2000, or greater than 3000, as determined by the plaque reduction neutralization test (PRNT).

Accordingly, certain aspects of the present disclosure relates to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart, and wherein the administration of the vaccine or immunogenic composition induces 28 days after the boost administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or of at least 20 Zika virus seronegative human subjects, which are at least 10 times, or at least 15 times, or at least 20 times, or at least 25 times higher than the geometric mean neutralizing antibody titers induced 28 days after the prime administration, as determined by the plaque reduction neutralization test (PRNT). The boost administration thus provides for very high geometric mean neutralizing antibody titers responsible for a long term protection

Accordingly, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a single dose or prime administration, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or of at least 20 Zika virus seronegative human subjects of greater than 10, or greater than 50, or greater than 100, or greater than 200, or greater than 250, as determined by the plaque reduction neutralization test (PRNT). The high geometric mean neutralizing antibody titers indicate an early onset of protection, which is beneficial in an outbreak situation or a traveler visiting an endemic area within a short period of time from the administration of the vaccine or immunogenic composition.

Within the meaning of this disclosure PRNT refers to Zika virus Neutralizing antibody titers in human subjects determined by a plaque reduction neutralization test (PRNT) as described previously (See Sun, W. et al. Protection of Rhesus monkeys against dengue virus challenge after tetravalent live attenuated dengue virus vaccination. J. Infect. Dis. 193, 1658-1665 (2006). Muthumani K, Griffin B D, Agarwal S, et al. In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine. NPJ Vaccines 2016; 1: 16021).

Within the meaning of this disclosure the GMT values in human subjects are in particular to be considered to be measured as the following. In the Zika PRNT assay, human serum was 2-fold serially diluted from 1:5 to 1:10,240 and mixed with an equal volume of diluted Zika virus (ZIKV) (PRVABC59) to obtain a final dilution of 1:10 to 1:20,480. Neutralization was allowed to proceed 20±2 hours at 2-8° C. after which the serum/virus mixture was used to inoculate Vero E6 cells. Virus adsorption was done at 37±2° C. with humidity and CO₂ for 60±10 minutes then a methylcellulose overlay was added. The infected cells were incubated at 37±2° C. with humidity and CO₂ for 72±2 hours. Plaques were visualized by using crystal violet staining and were counted using a CTL (Cellular Technology Limited) reader. Determination of the fifty percent neutralizing titer (PRNT₅₀) was based upon the percent reduction in viral plaques in the presence of serum compared to that of the virus control without serum and was calculated by linear regression. The titers represent the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques. Acceptance was assessed by evaluating the virus control (targeting ˜60 pfu/well), cell control, positive control (PRNT₅₀ of 173-658) and negative control (PRNT₅₀<10) tested in parallel with clinical samples. Individual samples and positive control results were accepted if the correlation coefficient of the titration curve generated by linear regression is ≥0.85. Additional acceptance criteria were based on the quality of the crystal violet stain and plaques generated for the plate or run. PRNT₅₀ results are reported down to the starting dilution of the assay (1:10). PRNT₅₀ results that are above the ULOQ will be repeated at a pre-dilution to generate a result within the quantifiable range of the assay. The result from the pre-diluted sample will be multiplied by the dilution factor to generate a final result.

Within the meaning of this disclosure seropositivity is defined as titer ≥10 as determined by the plaque reduction neutralization test (PRNT); Zika virus seronegative human subjects are subjects with a titer <10 as determined by the plaque reduction neutralization test (PRNT), Seroconversion is defined as: Zika virus seronegative human subjects (titer <10) have titer ≥10 post-vaccination as determined by the plaque reduction neutralization test (PRNT); Results <10 as determined by the plaque reduction neutralization test (PRNT) are assigned a titer of 5; Titers between 10 (limit of detection) and 26 (lower limit of quantification) as determined by the plaque reduction neutralization test (PRNT) are assigned a value of 13.

Flavivirus naïve human subjects for the present disclosure are defined to be human subjects without detectable serum antibodies against a panel of flaviviruses, as measured by a reactive antibody based assay (Luminex). Flavivirus screening assay is based on a luminex platform to simultaneously detect multiple target antigens in the same sample. This bead based assay is sensitive, specific and reproducible. For the present disclosure, the antigens targeted are Zika, Dengue, Yellow fever, JEV, USUV, SLEV and WNV. Due to cross reactivity among Flaviviruses, the current antigen set would help detect any prior Flavivirus exposure. References for luminex concept are: Dias D, Van Doren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, Boerckel P, Kessler J, Antonello J M, Green T, Brown M, Smith J, Chirmule N, Barr E, Jansen K U, Esser M T. 2005. Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18. Clin. Diagn. Lab. Immunol. 12:959-969 [PMC free article] [PubMed]. Ayouba A et al Development of a Sensitive and Specific Serological Assay Based on Luminex Technology for Detection of Antibodies to Zaire Ebola Virus. J Clin Microbiol. 2017 Dec. 28; 55(1):165-176. doi: 10.1128/JCM.01979-16.

Within the meaning of this disclosure endemic is defined as areas with risk of infection as defined by the Centers for Disease Control and Prevention, such as for example as of March 2018, namely: Asia: Bangladesh, Burma (Myanmar), Cambodia, India, Indonesia, Laos, Malaysia, Maldives, Pakistan, Philippines, Singapore, Thailand, Timor-Leste (East Timor), Vietnam. The Pacific Islands: Fiji, Marshall Islands, Papua New Guinea, Samoa, Solomon Islands, Tonga. The Caribbean: Anguilla; Antigua and Barbuda; Aruba; Barbados; Bonaire; British Virgin Islands; Cuba; Curacao; Dominica; Dominican Republic; Grenada; Haiti; Jamaica; Montserrat; the Commonwealth of Puerto Rico, a US territory; Saba; Saint Kitts and Nevis; Saint Lucia; Saint Martin; Saint Vincent and the Grenadines; Sint Eustatius; Sint Maarten; Trinidad and Tobago; Turks and Caicos Islands; US Virgin Islands. North America: Mexico Central America: Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, Panama South America: Argentina, Bolivia, Brazil, Colombia, Ecuador, French Guiana, Guyana, Paraguay, Peru, Suriname, Venezuela Africa: Angola, Benin, Burkina-Faso, Burundi, Cameroon, Cape Verde, Central African Republic, Chad, Congo (Congo-Brazzaville), Côte d'Ivoire, Democratic Republic of the Congo (Congo-Kinshasa), Equatorial Guinea, Gabon, Gambia, Ghana, Guinea, Guinea-Bissau, Kenya, Liberia, Mali, Niger, Nigeria, Rwanda, Senegal, Sierra Leone, South Sudan, Sudan, Tanzania, Togo, Uganda. These areas may change.

Accordingly, certain aspects of the present disclosure relate to a method for inducing an immune response in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the boost administration geometric mean neutralizing antibody titers in a human subject population of at least 20 flavivirus naïve human subjects and/or of at least 20 Zika virus seronegative human subjects of greater than 300, or greater than 500, or greater than 1000, or greater than 1500, or greater than 2000, or greater than 3000, or greater than 5000, or greater than 10,000, determined by the reporter virus particle neutralization assay (RVP).

Accordingly, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a single dose or prime administration, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or of at least 20 Zika virus seronegative human subjects of greater than 300, or greater than 500, greater than 1000, or greater than 2000, as determined by the reporter virus particle neutralization assay (RVP). The high geometric mean neutralizing antibody titers indicate an early onset of protection, which is beneficial in an outbreak situation or a traveler visiting an endemic area within a short period of time from the administration of the vaccine or immunogenic composition.

Within the meaning of this disclosure Reporter virus particle (RVP) neutralization assay refers to Zika Neutralizing antibody titers which were analyzed by titration of serum samples with a constant amount of Zika RVPs in Vero cells grown in 96-well plates. RVPs contained the prME proteins of Zika (strain SPH2012) and a Dengue-based Renilla luciferase reporter. Briefly, sera were heat inactivated at 56° C. for 30 min, diluted, and then incubated at 37° C. with RVPs. The serum/RVP mixture was then mixed with Vero cells and incubated for 72 hours at 37° C.±2° C./5% CO₂ before detection with luciferase substrate. Data was analyzed using JMP11 non-linear 4 parameter analysis, normalized to a positive tracking control and effective dose 50% (EC50) was reported.

Accordingly, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the boost administration a seroconversion rate of at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% in a population of at least 20 seronegative human subjects, as determined by the plaque reduction neutralization test (PRNT).

Accordingly, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as single dose or prime administration and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration a seroconversion rate of 25%, 30%, 40%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% in a population of at least 20 Zika virus seronegative human subjects, as determined by the plaque reduction neutralization test (PRNT). The high seroconversion rate after single dose or prime administration indicates an early onset of protection, which is beneficial in an outbreak situation or a traveler visiting an endemic area within a short period of time from the administration of the vaccine or immunogenic composition.

Accordingly, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as single dose or prime administration and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration a seropositivity rate of 25%, 30%, 40%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, or 90% in a population of at least 20 Flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects, as determined by the plaque reduction neutralization test (PRNT). The high seroconversion rate after single dose or prime administration indicates an early onset of protection, which is beneficial in an outbreak situation or a traveler visiting an endemic area within a short period of time from the administration of the vaccine or immunogenic composition.

Certain other aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising (one) antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a single dose or as multiple doses as e.g. in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the administration induces systemic side effects in less than 50% of a human subject population of at least 20 human subjects, in particular in a population of at least 20 flavivirus naïve human subjects or in particular in a population of at least 20 Zika virus seronegative human subjects. The above methods are to be understood to also relate to corresponding uses of a vaccine or immunogenic composition comprising (one) antigen from a Zika virus as disclosed herein for the manufacture of a medicament for the treating or preventing Zika virus infection.

Within the meaning of this disclosure “systemic side effects” are in particular fever, headache, fatigue, arthralgia, myalgia and malaise.

The above methods are to be understood to also relate to a vaccine or immunogenic composition comprising (one) antigen from a Zika virus as disclosed herein for use in treating or preventing Zika virus infection.

In some embodiments the administration is intramuscular or subcutaneous. In some embodiments the administration includes the administration of two doses of the vaccine or immunogenic composition as described herein (e.g. 10 μg purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein) given about 1 to about 16 weeks apart (first (prime) and a second (boost) administration). In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate, in particular as described herein.

The above aspects of the present disclosure relate to the vaccines or immunogenic compositions as described herein for use in treating or preventing Zika virus infection in a human subject in need thereof, for use in inducing an immune response in a human subject in need thereof and for use in preventing Zika virus disease in a human subject, fetus or newborn in need thereof. In some embodiments the administration is intramuscular or subcutaneous. In some embodiments the administration includes the administration of two doses of the vaccine or immunogenic composition as described herein (e.g. 10 μg purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein) given about 1 to about 16 weeks apart (first (prime) and a second (boost) administration). In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate, in particular as described herein.

The above aspects of the present disclosure relate to the use of the vaccines or immunogenic compositions as described herein in the manufacture of a medicament for treating or preventing Zika virus infection in a human subject in need thereof for inducing an immune response in a human subject in need thereof and in preventing Zika virus disease in a human subject, fetus or newborn in need thereof. In some embodiments the administration is intramuscular or subcutaneous. In some embodiments the administration includes the administration of two doses of the vaccine or immunogenic composition as described herein (e.g. 10 μg purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein) given about 1 to about 16 weeks apart (first (prime) and a second (boost) administration). In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate, in particular as described herein.

In some embodiments that may be combined with any of the preceding embodiments, the human subject is pregnant or intends to become pregnant or is a woman of childbearing potential.

In some embodiments that may be combined with any of the preceding embodiments, administration of the vaccine or immunogenic composition induces a protective immune response in the human subject. In some embodiments, the protective immune response induced in the human subject is greater than a protective immune response induced in a corresponding human subject administered a vaccine or immunogenic composition containing one or more antigens from a Zika virus lacking the at least one non-human cell adaptation mutation. In some embodiments that may be combined with any of the preceding embodiments, administration of the vaccine or immunogenic composition induces the generation of neutralizing antibodies to Zika virus in the human subject. In some embodiments, the concentration of neutralizing antibodies generated in the human subject is higher than a concentration of neutralizing antibodies generated in a corresponding human subject administered a vaccine or immunogenic composition comprising one or more antigens from a Zika virus lacking the at least one non-human cell adaptation mutation.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is administered by a route selected from subcutaneous administration, transcutaneous administration, intradermal administration, subdermal administration, intramuscular administration, peroral administration, intranasal administration, buccal administration, intraperitoneal administration, intravaginal administration, anal administration and/or intracranial administration. In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is administered one or more times. In some embodiments, the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration. In some embodiments, the second (boost) administration is administered at least 28 days after the first (prime) administration. In some embodiments the administration includes the administration of two doses of the vaccine or immunogenic composition as described herein (e.g. 10 μg purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein) given about 1 to about 16 weeks apart (first (prime) and a second (boost) administration). In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate.

In some embodiments that may be combined with any of the preceding embodiments, the virus preparation is mixed with an adjuvant. In some embodiments, the adjuvant is selected from aluminum salts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA), synthetic lipid A, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and/or Incomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the adjuvant is selected from alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and/or Alhydrogel 85. In some embodiments, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of one or more antigens in the virus preparation are adsorbed to the adjuvant.

In some embodiments that may be combined with any of the preceding embodiments, the at least one non-human cell adaptation mutation is in Zika virus NS1. In some embodiments, the at least one adaptation mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the at least one adaptation mutation is a Trp98Gly mutation. In some embodiments that may be combined with any of the preceding embodiments, the at least one adaptation mutation enhances genetic stability as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments that may be combined with any of the preceding embodiments, the at least one adaptation mutation enhances viral replication as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments that may be combined with any of the preceding embodiments, the Zika virus does not comprise a mutation in Envelope protein E (Env).

In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses is heterogeneous. In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses comprises a Zika virus clinical isolate. In some embodiments, the Zika virus clinical isolate is from strain PRVABC59. In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses comprises a Zika virus that has been previously passaged one or more times in cell culture. In some embodiments that may be combined with any of the preceding embodiments, the inoculum comprises human serum. In some embodiments that may be combined with any of the preceding embodiments, the inoculum comprises one or more adventitious agents. In some embodiments, the Zika virus clonal isolate is substantially free of the one or more adventitious agents.

In some embodiments that may be combined with any of the preceding embodiments, the methods further include one or more additional plaque purifications of the Zika virus clonal isolate. In some embodiments, the Zika virus clonal isolate is further plaque purified two or more times. In some embodiments that may be combined with any of the preceding embodiments, the methods further include passaging the Zika virus clonal isolate one or more times in cell culture. In some embodiments, the Zika virus clonal isolate is passaged two or more times.

In some embodiments that may be combined with any of the preceding embodiments, the methods further include formulating a vaccine or immunogenic composition comprising one or more antigens from the Zika virus clonal isolate. In some embodiments, the vaccine or immunogenic composition is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is a purified inactivated whole virus vaccine or immunogenic composition. In some embodiments, the Zika virus clonal isolate was chemically inactivated. In some embodiments, the Zika virus clonal isolate was chemically inactivated with one or more of a detergent, formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetyl ethyleneimine, methylene blue, and psoralen. In some embodiments, the Zika virus clonal isolate was chemically inactivated with formalin.

In some embodiments that may be combined with any of the preceding embodiments, the methods further include admixing the vaccine or immunogenic composition with an adjuvant. In some embodiments, the adjuvant is selected from aluminum salts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA), synthetic lipid A, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the adjuvant is selected from alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the one or more antigens are adsorbed to the adjuvant. In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition comprises 1 μg to about 40 μg of the purified inactivated whole virus, or 1 μg to about 30 μg of the purified inactivated whole virus, or 1 μg to about 20 μg of the purified inactivated whole virus, in particular 2 μg, or 5 μg, or 10 μg, or 15 μg or 20 μg or in particular 10 μg purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO: 1 as described herein. In some embodiments, the vaccine or immunogenic composition comprises 1 μg to about 30 μg of the purified inactivated whole virus, in particular 2 μg, 5 μg or 10 μg, or in particular 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59. In some embodiments, the vaccine or immunogenic composition comprises 1 μg to about 30 μg of the purified inactivated whole virus, in particular 2 μg, 5 μg or 10 μg, or in particular 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition comprises from about 0.1 μg Env to about 100 μg Env. In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate. In some embodiments, the vaccine or immunogenic composition is unadjuvanted.

In some embodiments that may be combined with any of the preceding embodiments, the Zika virus clonal isolate is a homogenous genetic population. In some embodiments, the Zika virus clonal isolate does not contain a mutation in Envelope protein E (Env). In some embodiments, the Zika virus clonal isolate contains at least one mutation. In some embodiments, the at least one mutation is in Zika virus Non-structural protein 1 (NS1). In some embodiments, the at least one mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the at least one mutation is a Trp98Gly mutation. In some embodiments the at least one mutation is not in Envelope protein E (Env). In some embodiments, the at least one mutation enhances genetic stability as compared to a Zika virus lacking the at least one mutation. In some embodiments, the at least one mutation enhances viral replication as compared to a Zika virus lacking the at least one mutation.

Other aspects of the present disclosure relate to a vaccine or immunogenic composition containing one or more antigens from a plaque purified clonal Zika virus isolate. In some embodiments, the plaque purified clonal Zika virus isolate was plaque purified from cells contacted with an inoculum comprising a population of Zika viruses. In some embodiments, the cells are non-human cells. In some embodiments, the cells are insect cells. In some embodiments, the insect cells are mosquito cells. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are monkey cells. In some embodiments, the monkey cells are from a Vero cell line. In some embodiments, the Vero cell line is a WHO Vero 10-87 cell line.

In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses was heterogeneous. In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses comprised a Zika virus clinical isolate. In some embodiments, the Zika virus clinical isolate is from strain PRVABC59. In some embodiments that may be combined with any of the preceding embodiments, the population of Zika viruses comprised a Zika virus that had been previously passaged one or more times in cell culture. In some embodiments that may be combined with any of the preceding embodiments, the inoculum comprised human serum. In some embodiments that may be combined with any of the preceding embodiments, the inoculum comprised one or more adventitious agents. In some embodiments, the plaque purified clonal Zika virus isolate is substantially free of the one or more adventitious agents.

In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate is modified as compared to a wild-type Zika virus. In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate is a homogenous genetic population. In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate does not include a mutation in Envelope protein E (Env). In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate comprises at least one mutation. In some embodiments, the at least one mutation is in Zika virus Non-structural protein 1 (NS1). In some embodiments, the at least one mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. In some embodiments, the at least one mutation is a Trp98Gly mutation. In some embodiments, the at least one mutation is not in Zika virus Envelope protein E (Env). In some embodiments, the at least one mutation enhances genetic stability as compared to a Zika virus lacking the at least one mutation. In some embodiments, the at least one mutation enhances viral replication as compared to a Zika virus lacking the at least one mutation. In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate is an African lineage virus or an Asian lineage virus. In some embodiments, the plaque purified clonal Zika virus isolate is an Asian lineage virus.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is an inactivated whole virus vaccine or immunogenic composition. In some embodiments that may be combined with any of the preceding embodiments, the plaque purified clonal Zika virus isolate was chemically inactivated. In some embodiments, the plaque purified Zika virus was chemically inactivated with one or more of a detergent, formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetyl ethyleneimine, methylene blue, and psoralen. In some embodiments, the plaque purified clonal Zika virus isolate was chemically inactivated with formalin.

In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition further comprises an adjuvant. In some embodiments, the adjuvant is selected from aluminum salts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA), synthetic lipid A, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the adjuvant is selected from alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the one or more antigens are adsorbed to the adjuvant. In some embodiments that may be combined with any of the preceding embodiments, the vaccine or immunogenic composition contains from 0.1 μg to about 25 μg of the purified inactivated whole virus, such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 as described herein, in particular 2 μg, 5 μg or 10 μg, or in particular 10 μg purified inactivated whole virus or about 0.1 μg Env to about 100 μg Env. In certain such embodiments the Zika virus is a plaque purified clonal Zika virus isolate. In some embodiments, the vaccine or immunogenic composition is unadjuvanted.

It is to be understood that one, some, or all of the properties of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to one of skill in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows bright field microscopy images of Vero cell monolayers mock infected (top) or infected with ZIKAV strain PRVABC59 (bottom).

FIG. 2 shows growth kinetics of ZIKAV PRVABC59 P1 on Vero cell monolayers, as determined by TCID50.

FIG. 3 shows potency assay testing (TCID50) of Zika virus PRVABC59 P5 clones a-f.

FIG. 4 shows bright-field microscopy images depicting the cytopathic effect (CPE) of growth of Zika virus PRVABC59 P6 clones a-f on Vero cell monolayers.

FIG. 5 shows potency assay testing (TCID50) of Zika virus PRVABC59 P6 clones a-f

FIG. 6 shows an amino acid sequence alignment comparing the envelope glycoprotein sequence of Zika virus near residue 330 from Zika virus strains PRVABC59 P6e (SEQ ID NO: 8) and PRVABC59 (SEQ ID NO: 9) with several other flaviviruses (WNV (SEQ ID NO: 10); JEV (SEQ ID NO: 11); SLEV (SEQ ID NO: 12); YFV (SEQ ID NO: 13); DENY 1 16007 (SEQ ID NO: 14); DENY 2 16681 (SEQ ID NO: 15); DENY 3 16562 (SEQ ID NO: 16); and DENY 4 1036 (SEQ ID NO: 17)).

FIG. 7 shows an amino acid sequence alignment comparing the NS1 protein sequence of Zika virus near residue 98 from Zika virus strains PRVABC59 P6e (SEQ ID NO: 18) and PRVABC59 (SEQ ID NO: 19) with several other flaviviruses (WNV (SEQ ID NO: 20); JEV (SEQ ID NO: 21); SLEV (SEQ ID NO: 22); YFV (SEQ ID NO: 23); DENY 1 16007 (SEQ ID NO: 24); DENY 2 16681 (SEQ ID NO: 25); DENY 3 16562 (SEQ ID NO: 26); and DENY 4 1036 (SEQ ID NO: 27)).

FIG. 8 shows the plaque phenotype of ZIKAV PRVABC59 P6 virus clones a-f compared to ZIKAV PRVABC59 P1 virus.

FIG. 9 shows the mean plaque size of ZIKAV PRVABC59 P6 virus clones compared to ZIKAV PRVABC59 P1 virus.

FIG. 10 shows the growth kinetics of ZIKAV PRVABC59 P6 clones a-f in Vero cells under serum-free growth conditions.

FIG. 11 shows a schematic of the steps taken to prepare PRVABC59 P6b and P6e formulated drug product for the immunization experiments.

FIG. 12A shows the schedule of dosing of CD-1 mice with vaccine formulations derived from the ZIKAV PRVABC59 P6b and P6e clones. PBS was used as placebo.

FIG. 12B shows the serum ZIKAV neutralizing antibody titers of CD-1 mice immunized as described in FIG. 12A using vaccine formulations derived from ZIKAV PRVABC59 P6b and P6e clones. ZIKAV neutralizing antibody titers were determined by Reporter Virus Particle (RVP) neutralization assay. Solid lines represent the geometric mean of a group. The limit of detection (1.93 log₁₀) is represented by a dashed line.

FIG. 13A shows the schedule of dosing of AG129 mice with vaccine formulations derived from the ZIKAV PRVABC59 P6b and P6e clones. PBS was used as a placebo.

FIG. 13B shows the serum ZIKAV neutralizing antibody titers of AG129 mice immunized as described in FIG. 13A using vaccine formulations derived from ZIKAV PRVABC59 P6b and P6e clones. Solid lines represent the geometric mean of a group. The limit of detection (1.30 log₁₀) is represented by a dashed line. Animals with no detectable titer (<1.30) were assigned a titer of 0.5.

FIG. 14 shows the mean weight of AG129 test groups post-challenge, represented as a percentage of starting weight. Error bars represent standard deviation.

FIG. 15 shows the serum viremia of individual AG129 mice two days post-challenge, reported as PFU/mL. Solid lines represent the mean of a group. The limit of detection (2.0 log₁₀) is represented by a dashed line.

FIG. 16 shows the survival analysis of AG129 test groups post-challenge.

FIG. 17 shows the pre-challenge serum circulating ZIKAV neutralizing antibody (Nab) titers following passive transfer of pooled sera from vaccinated and challenged AG129 mice.

FIG. 18 shows the mean body weight of passive transfer and control mice challenged with Zika virus.

FIG. 19 shows the serum viremia of individual AG129 mice three days post-challenge, reported as PFU/mL.

FIG. 20 shows the survival analysis of passive transfer and control mice challenged with Zika virus.

FIG. 21 shows the correlation between ZIKAV neutralizing antibody titers and viremia observed in passive transfer mice.

FIG. 22 shows the survival analysis of AG129 mice after challenge with preMVS stocks of P6a and P6e using a Kaplan Meier survival curve.

FIG. 23 shows the mean body weight as expressed in percentage of starting weight at time of invention after challenge with preMVS stocks of P6a and P6e. The dashed line represents 100% of starting weight for reference.

FIG. 24 shows the serum viremia of individual AG129 mice three days post-challenge with preMVS stocks of P6a and P6e, reported as PFU/mL. The dashed line represents the limit of detection of the assay.

FIG. 25 shows compiled kinetics of inactivation data. Data compares infectious potency (TCID50) to RNA copy, and completeness of inactivation (COI) for samples from the four toxicology lots. These data indicate that the sensitivity of the COI assay is greater than TCID50.

FIG. 26 shows a comparison of C6/36 and Vero sensitivity in the assay as demonstrated with an input virus titer of 0.31 TCID50.

FIG. 27 shows a logistic regression analysis of CPE vs. log TCID50 using C6/36 cells site that include 99% confidence intervals around a target value of 0.01 TCID50/well (−2 log TCID50/well); the model predicts 0.85% of wells will be positive.

FIG. 28 shows chromatograms of PBS (a) and PBS solutions containing 0.049 μg/mL (b), 0.098 μg/mL (c), 0.196 μg/mL (d), 0.491 μg/mL (e), 0.982 μg/mL (f), and 1.964 μg/mL (g) formaldehyde.

FIG. 29 shows a summary of the Clinical Study Design for Example 6.

FIG. 30 shows the Geometric Mean Titers (GMTs) determined using PRNT of the Human subjects in Example 6

FIG. 31 shows the percentage of human subjects achieving seroconversion determined using PRNT at Day 29 (day 28 after prime dose) and Day 57 (28 days after boost dose) of the study described in Example 6.

FIG. 32 shows the plot of the percentage of human subjects achieving a particular Geometric Mean Titer (determined using PRNT) on day 29 (day 28 after prime dose) of the study described in Example 6.

FIG. 33 shows the plot of the percentage of human subjects achieving a particular Geometric Mean Titer (determined using PRNT) on day 57 (day 56 after prime dose) of the study described in Example 6.

DETAILED DESCRIPTION General Techniques

The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Antibodies, A Laboratory Manual (Harlow and Lane, eds. (1988), and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology (J. M. Walker, ed. Humana Press (1983)); Cell Biology: A Laboratory Notebook (J. E. Celis, ed., Academic Press (1998)) Academic Press; Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, eds. Plenum Press (1998)); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wiley and Sons (1993-8)); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory (1987)); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., Springer (1994)); Current Protocols in Immunology (J. E. Coligan et al., eds., Wiley (1991)); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, (1997)); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, (1988-1989)); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, (2000)); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, (1999)); and The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, (1995)).

Zika Virus

Certain aspects of the present disclosure relate to at least one Zika virus (e.g., a Zika virus clonal isolate, a Zika virus purified by the methods described herein, a Zika virus comprising one or more non-human cell adaptation mutations, etc.) that may be useful in vaccines and/or immunogenic compositions including, without limitation, purified viruses, inactivated viruses, attenuated viruses, recombinant viruses, or purified and/or recombinant viral proteins for subunit vaccines.

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated from a sentinel rhesus monkey in the Zika Forest in Uganda in 1947. Since that time, isolations have been made from humans in both Africa and Asia, and more recently, the Americas. ZIKV is found in two (possibly three) lineages: an African lineage (possibly separate East and West African lineages) and an Asian lineage. Accordingly, examples of suitable Zika viruses of the present disclosure include, without limitation, viruses from the African and/or Asian lineages. In some embodiments, the Zika virus is an African lineage virus. In some embodiments, the Zika virus is an Asian lineage virus. Additionally, multiple strains within the African and Asian lineages of Zika virus have been previously identified. Any one or more suitable strains of Zika virus known in the art may be used in the present disclosure, including, for examples, strains Mr 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD 7117, ArD 9957, ArD 30101, ArD 30156, ArD 30332, HD 78788, ArD 127707, ArD 127710, ArD 127984, ArD 127988, ArD 127994, ArD 128000, ArD 132912, 132915, ArD 141170, ArD 142623, ArD 149917, ArD 149810, ArD 149938, ArD 157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA 27101, ArA 27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506/96, ArA 975-99, Ara 982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria68, Malaysia66, Kedougou84, Suriname, MR1429, PRVABC59, ECMN2007, DakAr41524, H/PF/2013, R103451, 103344, 8375, JMB-185, ZIKV/H, sapiens/Brazil/Natal/2015, SPH2015, ZIKV/Hu/Chiba/S36/2016, and/or Cuba2017. In some embodiments, strain PRVABC59 is used in the present disclosure.

In some embodiments, an example of a Zika virus genome sequence is set forth below as SEQ ID NO: 2:

    1 gttgttgatc tgtgtgaatc agactgcgac agttcgagtt tgaagcgaaa gctagcaaca     61 gtatcaacag gttttatttt ggatttggaa acgagagttt ctggtcatga aaaacccaaa    121 aaagaaatcc ggaggattcc ggattgtcaa tatgctaaaa cgcggagtag cccgtgtgag    181 cccctttggg ggcttgaaga ggctgccagc cggacttctg ctgggtcatg ggcccatcag    241 gatggtcttg gcgattctag cctttttgag attcacggca atcaagccat cactgggtct    301 catcaataga tggggttcag tggggaaaaa agaggctatg gaaacaataa agaagttcaa    361 gaaagatctg gctgccatgc tgagaataat caatgctagg aaggagaaga agagacgagg    421 cgcagatact agtgtcggaa ttgttggcct cctgctgacc acagctatgg cagcggaggt    481 cactagacgt gggagtgcat actatatgta cttggacaga aacgatgctg gggaggccat    541 atcttttcca accacattgg ggatgaataa gtgttatata cagatcatgg atcttggaca    601 catgtgtgat gccaccatga gctatgaatg ccctatgctg gatgaggggg tggaaccaga    661 tgacgtcgat tgttggtgca acacgacgtc aacttgggtt gtgtacggaa cctgccatca    721 caaaaaaggt gaagcacgga gatctagaag agctgtgacg ctcccctccc attccaccag    781 gaagctgcaa acgcggtcgc aaacctggtt ggaatcaaga gaatacacaa agcacttgat    841 tagagtcgaa aattggatat tcaggaaccc tggcttcgcg ttagcagcag ctgccatcgc    901 ttggcttttg ggaagctcaa cgagccaaaa agtcatatac ttggtcatga tactgctgat    961 tgccccggca tacagcatca ggtgcatagg agtcagcaat agggactttg tggaaggtat   1021 gtcaggtggg acttgggttg atgttgtctt ggaacatgga ggttgtgtca ccgtaatggc   1081 acaggacaaa ccgactgtcg acatagagct ggttacaaca acagtcagca acatggcgga   1141 ggtaagatcc tactgctatg aggcatcaat atcagacatg gcttctgaca gccgctgccc   1201 aacacaaggt gaagcctacc ttgacaagca atcagacact caatatgtct gcaaaagaac   1261 gttagtggac agaggctggg gaaatggatg tggacttttt ggcaaaggga gcctggtgac   1321 atgcgctaag tttgcatgct ccaagaaaat gaccgggaag agcatccagc cagagaatct   1381 ggagtaccgg ataatgctgt cagttcatgg ctcccagcac agtgggatga tcgttaatga   1441 cacaggacat gaaactgatg agaatagagc gaaagttgag ataacgccca attcaccgag   1501 agccgaagcc accctggggg gttttggaag cctaggactt gattgtgaac cgaggacagg   1561 ccttgacttt tcagatttgt attacttgac tatgaataac aagcactggt tggttcacaa   1621 ggagtggttc cacgacattc cattaccttg gcacgctggg gcagacaccg gaactccaca   1681 ctggaacaac aaagaagcac tggtagagtt caaggacgca catgccaaaa ggcaaactgt   1741 cgtggttcta gggagtcaag aaggagcagt tcacacggcc cttgctggag ctctggaggc   1801 tgagatggat ggtgcaaagg gaaggctgtc ctctggccac ttgaaatgtc gcctgaaaat   1861 ggataaactt agattgaagg gcgtgtcata ctccttgtgt actgcagcgt tcacattcac   1921 caagatcccg gctgaaacac tgcacgggac agtcacagtg gaggtacagt acgcagggac   1981 agatggacct tgcaaggttc cagctcagat ggcggtggac atgcaaactc tgaccccagt   2041 tgggaggttg ataaccgcta accccgtaat cactgaaagc actgagaact ctaagatgat   2101 gctggaactt gatccaccat ttggggactc ttacattgtc ataggagtcg gggagaagaa   2161 gatcacccac cactggcaca ggagtggcag caccattgga aaagcatttg aagccactgt   2221 gagaggtgcc aagagaatgg cagtcttggg agacacagcc tgggactttg gatcagttgg   2281 aggcgctctc aactcattgg gcaagggcat ccatcaaatt tttggagcag ctttcaaatc   2341 attgtttgga ggaatgtcct ggttctcaca aattctcatt ggaacgttgc tgatgtggtt   2401 gggtctgaac acaaagaatg gatctatttc ccttatgtgc ttggccttag ggggagtgtt   2461 gatcttctta tccacagccg tctctgctga tgtggggtgc tcggtggact tctcaaagaa   2521 ggagacgaga tgcggtacag gggtgttcgt ctataacgac gttgaagcct ggagggacag   2581 gtacaagtac catcctgact ccccccgtag attggcagca gcagtcaagc aagcctggga   2641 agatggtatc tgcgggatct cctctgtttc aagaatggaa aacatcatgt ggagatcagt   2701 agaaggggag ctcaacgcaa tcctggaaga gaatggagtt caactgacgg tcgttgtggg   2761 atctgtaaaa aaccccatgt ggagaggtcc acagagattg cccgtgcctg tgaacgagct   2821 gccccacggc tggaaggctt gggggaaatc gtatttcgtc agagcagcaa agacaaataa   2881 cagctttgtc gtggatggtg acacactgaa ggaatgccca ctcaaacata gagcatggaa   2941 cagctttctt gtggaggatc atgggttcgg ggtatttcac actagtgtct ggctcaaggt   3001 tagagaagat tattcattag agtgtgatcc agccgttatt ggaacagctg ttaagggaaa   3061 ggaggctgta cacagtgatc taggctactg gattgagagt gagaagaatg acacatggag   3121 gctgaagagg gcccatctga tcgagatgaa aacatgtgaa tggccaaagt cccacacatt   3181 gtggacagat ggaatagaag agagtgatct gatcataccc aagtctttag ctgggccact   3241 cagccatcac aataccagag agggctacag gacccaaatg aaagggccat ggcacagtga   3301 agagcttgaa attcggtttg aggaatgccc aggcactaag gtccacgtgg aggaaacatg   3361 tggaacaaga ggaccatctc tgagatcaac cactgcaagc ggaagggtga tcgaggaatg   3421 gtgctgcagg gagtgcacaa tgcccccact gtcgttccgg gctaaagatg gctgttggta   3481 tggaatggag ataaggccca ggaaagaacc agaaagcaac ttagtaaggt caatggtgac   3541 tgcaggatca actgatcaca tggaccactt ctcccttgga gtgcttgtga tcctgctcat   3601 ggtgcaggaa gggctgaaga agagaatgac cacaaagatc atcataagca catcaatggc   3661 agtgctggta gctatgatcc tgggaggatt ttcaatgagt gacctggcta agcttgcaat   3721 tttgatgggt gccaccttcg cggaaatgaa cactggagga gatgtagctc atctggcgct   3781 gatagcggca ttcaaagtca gaccagcgtt gctggtatct ttcatcttca gagctaattg   3841 gacaccccgt gaaagcatgc tgctggcctt ggcctcgtgt cttttgcaaa ctgcgatctc   3901 cgccttggaa ggcgacctga tggttctcat caatggtttt gctttggcct ggttggcaat   3961 acgagcgatg gttgttccac gcactgataa catcaccttg gcaatcctgg ctgctctgac   4021 accactggcc cggggcacac tgcttgtggc gtggagagca ggccttgcta cttgcggggg   4081 gtttatgctc ctctctctga agggaaaagg cagtgtgaag aagaacttac catttgtcat   4141 ggccctggga ctaaccgctg tgaggctggt cgaccccatc aacgtggtgg gactgctgtt   4201 gctcacaagg agtgggaagc ggagctggcc ccctagcgaa gtactcacag ctgttggcct   4261 gatatgcgca ttggctggag ggttcgccaa ggcagatata gagatggctg ggcccatggc   4321 cgcggtcggt ctgctaattg tcagttacgt ggtctcagga aagagtgtgg acatgtacat   4381 tgaaagagca ggtgacatca catgggaaaa agatgcggaa gtcactggaa acagtccccg   4441 gctcgatgtg gcgctagatg agagtggtga tttctccctg gtggaggatg acggtccccc   4501 catgagagag atcatactca aggtggtcct gatgaccatc tgtggcatga acccaatagc   4561 catacccttt gcagctggag cgtggtacgt atacgtgaag actggaaaaa ggagtggtgc   4621 tctatgggat gtgcctgctc ccaaggaagt aaaaaagggg gagaccacag atggagtgta   4681 cagagtaatg actcgtagac tgctaggttc aacacaagtt ggagtgggag ttatgcaaga   4741 gggggtcttt cacactatgt ggcacgtcac aaaaggatcc gcgctgagaa gcggtgaagg   4801 gagacttgat ccatactggg gagatgtcaa gcaggatctg gtgtcatact gtggtccatg   4861 gaagctagat gccgcctggg atgggcacag cgaggtgcag ctcttggccg tgccccccgg   4921 agagagagcg aggaacatcc agactctgcc cggaatattt aagacaaagg atggggacat   4981 tggagcggtt gcgctggatt acccagcagg aacttcagga tctccaatcc tagacaagtg   5041 tgggagagtg ataggacttt atggcaatgg ggtcgtgatc aaaaacggga gttatgttag   5101 tgccatcacc caagggagga gggaggaaga gactcctgtt gagtgcttcg agccctcgat   5161 gctgaagaag aagcagctaa ctgtcttaga cttgcatcct ggagctggga aaaccaggag   5221 agttcttcct gaaatagtcc gtgaagccat aaaaacaaga ctccgtactg tgatcttagc   5281 tccaaccagg gttgtcgctg ctgaaatgga ggaggccctt agagggcttc cagtgcgtta   5341 tatgacaaca gcagtcaatg tcacccactc tggaacagaa atcgtcgact taatgtgcca   5401 tgccaccttc acttcacgtc tactacagcc aatcagagtc cccaactata atctgtatat   5461 tatggatgag gcccacttca cagatccctc aagtatagca gcaagaggat acatttcaac   5521 aagggttgag atgggcgagg cggctgccat cttcatgacc gccacgccac caggaacccg   5581 tgacgcattt ccggactcca actcaccaat tatggacacc gaagtggaag tcccagagag   5641 agcctggagc tcaggctttg attgggtgac ggatcattct ggaaaaacag tttggtttgt   5701 tccaagcgtg aggaacggca atgagatcgc agcttgtctg acaaaggctg gaaaacgggt   5761 catacagctc agcagaaaga cttttgagac agagttccag aaaacaaaac atcaagagtg   5821 ggactttgtc gtgacaactg acatttcaga gatgggcgcc aactttaaag ctgaccgtgt   5881 catagattcc aggagatgcc taaagccggt catacttgat ggcgagagag tcattctggc   5941 tggacccatg cctgtcacac atgccagcgc tgcccagagg agggggcgca taggcaggaa   6001 tcccaacaaa cctggagatg agtatctgta tggaggtggg tgcgcagaga ctgacgaaga   6061 ccatgcacac tggcttgaag caagaatgct ccttgacaat atttacctcc aagatggcct   6121 catagcctcg ctctatcgac ctgaggccga caaagtagca gccattgagg gagagttcaa   6181 gcttaggacg gagcaaagga agacctttgt ggaactcatg aaaagaggag atcttcctgt   6241 ttggctggcc tatcaggttg catctgccgg aataacctac acagatagaa gatggtgctt   6301 tgatggcacg accaacaaca ccataatgga agacagtgtg ccggcagagg tgtggaccag   6361 acacggagag aaaagagtgc tcaaaccgag gtggatggac gccagagttt gttcagatca   6421 tgcggccctg aagtcattca aggagtttgc cgctgggaaa agaggagcgg cttttggagt   6481 gatggaagcc ctgggaacac tgccaggaca catgacagag agattccagg aagccattga   6541 caacctcgct gtgctcatgc gggcagagac tggaagcagg ccttacaaag ccgcggcggc   6601 ccaattgccg gagaccctag agaccataat gcttttgggg ttgctgggaa cagtctcgct   6661 gggaatcttc ttcgtcttga tgaggaacaa gggcataggg aagatgggct ttggaatggt   6721 gactcttggg gccagcgcat ggctcatgtg gctctcggaa attgagccag ccagaattgc   6781 atgtgtcctc attgttgtgt tcctattgct ggtggtgctc atacctgagc cagaaaagca   6841 aagatctccc caggacaacc aaatggcaat catcatcatg gtagcagtag gtcttctggg   6901 cttgattacc gccaatgaac tcggatggtt ggagagaaca aagagtgacc taagccatct   6961 aatgggaagg agagaggagg gggcaaccat aggattctca atggacattg acctgcggcc   7021 agcctcagct tgggccatct atgctgcctt gacaactttc attaccccag ccgtccaaca   7081 tgcagtgacc acctcataca acaactactc cttaatggcg atggccacgc aagctggagt   7141 gttgtttggc atgggcaaag ggatgccatt ctacgcatgg gactttggag tcccgctgct   7201 aatgataggt tgctactcac aattaacacc cctgacccta atagtggcca tcattttgct   7261 cgtggcgcac tacatgtact tgatcccagg gctgcaggca gcagctgcgc gtgctgccca   7321 gaagagaacg gcagctggca tcatgaagaa ccctgttgtg gatggaatag tggtgactga   7381 cattgacaca atgacaattg acccccaagt ggagaaaaag atgggacagg tgctactcat   7441 agcagtagcc gtctccagcg ccatactgtc gcggaccgcc tgggggtggg gggaggctgg   7501 ggctctgatc acagccgcaa cttccacttt gtgggaaggc tctccgaaca agtactggaa   7561 ctcctctaca gccacttcac tgtgtaacat ttttagggga agttacttgg ctggagcttc   7621 tctaatctac acagtaacaa gaaacgctgg cttggtcaag agacgtgggg gtggaacagg   7681 agagaccctg ggagagaaat ggaaggcccg cttgaaccag atgtcggccc tggagttcta   7741 ctcctacaaa aagtcaggca tcaccgaggt gtgcagagaa gaggcccgcc gcgccctcaa   7801 ggacggtgtg gcaacgggag gccatgctgt gtcccgagga agtgcaaagc tgagatggtt   7861 ggtggagcgg ggatacctgc agccctatgg aaaggtcatt gatcttggat gtggcagagg   7921 gggctggagt tactacgtcg ccaccatccg caaagttcaa gaagtgaaag gatacacaaa   7981 aggaggccct ggtcatgaag aacccgtgtt ggtgcaaagc tatgggtgga acatagtccg   8041 tcttaagagt ggggtggacg tctttcatat ggcggctgag ccgtgtgaca cgttgctgtg   8101 tgacataggt gagtcatcat ctagtcctga agtggaagaa gcacggacgc tcagagtcct   8161 ctccatggtg ggggattggc ttgaaaaaag accaggagcc ttttgtataa aagtgttgtg   8221 cccatacacc agcactatga tggaaaccct ggagcgactg cagcgtaggt atgggggagg   8281 actggtcaga gtgccactct cccgcaactc tacacatgag atgtactggg tctctggagc   8341 gaaaagcaac accataaaaa gtgtgtccac cacgagccag ctcctcttgg ggcgcatgga   8401 cgggcctagg aggccagtga aatatgagga ggatgtgaat ctcggctctg gcacgcgggc   8461 tgtggtaagc tgcgctgaag ctcccaacat gaagatcatt ggtaaccgca ttgaaaggat   8521 ccgcagtgag cacgcggaaa cgtggttctt tgacgagaac cacccatata ggacatgggc   8581 ttaccatgga agctatgagg cccccacaca agggtcagcg tcctctctaa taaacggggt   8641 tgtcaggctc ctgtcaaaac cctgggatgt ggtgactgga gtcacaggaa tagccatgac   8701 cgacaccaca ccgtatggtc agcaaagagt tttcaaggaa aaagtggaca ctagggtgcc   8761 agacccccaa gaaggcactc gtcaggttat gagcatggtc tcttcctggt tgtggaaaga   8821 gctaggcaaa cacaaacggc cacgagtctg caccaaagaa gagttcatca acaaggttcg   8881 tagcaatgca gcattagggg caatatttga agaggaaaaa gagtggaaga ctgcagtgga   8941 agctgtgaac gatccaaggt tctgggctct agtggacaag gaaagagagc accacctgag   9001 aggagagtgc cagagctgtg tgtacaacat gatgggaaaa agagaaaaga aacaagggga   9061 atttggaaag gccaagggca gccgcgccat ctggtatatg tggctagggg ctagatttct   9121 agagttcgaa gcccttggat tcttgaacga ggatcactgg atggggagag agaactcagg   9181 aggtggtgtt gaagggctgg gattacaaag actcggatat gtcctagaag agatgagtcg   9241 tataccagga ggaaggatgt atgcagatga cactgctggc tgggacaccc gcattagcag   9301 gtttgatctg gagaatgaag ctctaatcac caaccaaatg gagaaagggc acagggcctt   9361 ggcattggcc ataatcaagt acacatacca aaacaaagtg gtaaaggtcc ttagaccagc   9421 tgaaaaaggg aaaacagtta tggacattat ttcgagacaa gaccaaaggg ggagcggaca   9481 agttgtcact tacgctctta acacatttac caacctagtg gtgcaactca ttcggaatat   9541 ggaggctgag gaagttctag agatgcaaga cttgtggctg ctgcggaggt cagagaaagt   9601 gaccaactgg ttgcagagca acggatggga taggctcaaa cgaatggcag tcagtggaga   9661 tgattgcgtt gtgaagccaa ttgatgatag gtttgcacat gccctcaggt tcttgaatga   9721 tatgggaaaa gttaggaagg acacacaaga gtggaaaccc tcaactggat gggacaactg   9781 ggaagaagtt ccgttttgct cccaccactt caacaagctc catctcaagg acgggaggtc   9841 cattgtggtt ccctgccgcc accaagatga actgattggc cgggcccgcg tctctccagg   9901 ggcgggatgg agcatccggg agactgcttg cctagcaaaa tcatatgcgc aaatgtggca   9961 gctcctttat ttccacagaa gggacctccg actgatggcc aatgccattt gttcatctgt  10021 gccagttgac tgggttccaa ctgggagaac tacctggtca atccatggaa agggagaatg  10081 gatgaccact gaagacatgc ttgtggtgtg gaacagagtg tggattgagg agaacgacca  10141 catggaagac aagaccccag ttacgaaatg gacagacatt ccctatttgg gaaaaaggga  10201 agacttgtgg tgtggatctc tcatagggca cagaccgcgc accacctggg ctgagaacat  10261 taaaaacaca gtcaacatgg tgcgcaggat cataggtgat gaagaaaagt acatggacta  10321 cctatccacc caagttcgct acttgggtga agaagggtct acacctggag tgctgtaagc  10381 accaatctta atgttgtcag gcctgctagt cagccacagc ttggggaaag ctgtgcagcc  10441 tgtgaccccc ccaggagaag ctgggaaacc aagcctatag tcaggccgag aacgccatgg  10501 cacggaagaa gccatgctgc ctgtgagccc ctcagaggac actgagtcaa aaaaccccac  10561 gcgcttggag gcgcaggatg ggaaaagaag gtggcgacct tccccaccct tcaatctggg  10621 gcctgaactg gagatcagct gtggatctcc agaagaggga ctagtggtta gagga 

In some embodiments, the Zika virus may comprise the genome sequence of GenBank Accession number KU501215.1. In some embodiments, the Zika virus is from strain PRVABC59. In some embodiments the genome sequence of GenBank Accession number KU501215.1 comprises the sequence of SEQ ID NO: 2. In some embodiments, the Zika virus may comprise a genomic sequence that has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2.

In some embodiments, the Zika virus may comprise at least one polypeptide encoded by the sequence of SEQ ID NO: 2. In some embodiments, the Zika virus may comprise at least one polypeptide having an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence encoded by the sequence of SEQ ID NO: 2.

Accordingly, in some embodiments, Zika viruses of the present disclosure may be used in any of the vaccines and/or immunogenic compositions disclosed herein. For example, Zika viruses of the present disclosure may be used to provide one or more antigens useful for treating or preventing Zika virus infection in a human subject in need thereof and/or for inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof.

Viral Antigens

In some embodiments, the present disclosure relates to one or more antigens from any Zika virus described herein useful in vaccines and/or immunogenic compositions including, without limitation, purified viruses, inactivated viruses, attenuated viruses, recombinant viruses, or purified and/or recombinant viral proteins for subunit vaccines. In some embodiments, the vaccines and/or immunogenic compositions include inactivated whole viruses.

Antigens of the present disclosure may be any substance capable of eliciting an immune response. Examples of suitable antigens include, but are not limited to, whole virus, attenuated virus, inactivated virus, proteins, polypeptides (including active proteins and individual polypeptide epitopes within proteins), glycopolypeptides, lipopolypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates.

Antigens of the present disclosure may be from any Zika virus (e.g., a Zika virus clonal isolate) produced from one or more cells in cell culture (e.g., via plaque purification). Any suitable cells known in the art for producing Zika virus may be used, including, for example, insect cells (e.g., mosquito cells such as CCL-125 cells, Aag-2 cells, RML-12 cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t. GRIP-1 cells, A.t. GRIP-2 cells, A.t. GRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells, NIID-CTR cells, TRA-171, cells, and additional cells or cell lines from mosquito species such as Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and/or Toxorhynchites amboinensis), and mammalian cells (e.g., VERO cells (from monkey kidneys), LLC-MK2 cells (from monkey kidneys), MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219 as described in WO97/37001) cells, BHK21-F cells, HKCC cells, or Chinese hamster ovary cells (CHO cells). In some embodiments, antigens of the present disclosure are from a Zika virus (e.g., a Zika virus clonal isolate) produced from a non-human cell (e.g., via plaque purification). In some embodiments, antigens of the present disclosure are from a Zika virus (e.g., a Zika virus clonal isolate) produced from an insect cell (e.g., via plaque purification). In some embodiments, antigens of the present disclosure are from a Zika virus (e.g., a Zika virus clonal isolate) produced from a mosquito cell (e.g., via plaque purification). In some embodiments, antigens of the present disclosure are from a Zika virus (e.g., a Zika virus clonal isolate) produced from a mammalian cell (e.g., via plaque purification). In some embodiments, antigens of the present disclosure are from a Zika virus (e.g., a Zika virus clonal isolate) produced from a VERO cell (e.g., via plaque purification). Methods of purifying a virus by performing plaque purification are known to one of ordinary skill in the art (See e.g., Example 1 below).

Antigens of the present disclosure may include at least one non-human cell adaptation mutation. Adaptation mutations may be generated by adapting a virus to growth in a particular cell line. For example, a cell may be transfected or electroporated with a virus, RNA transcribed from a virus (e.g., an infectious virus, or infectious clone), and/or RNA purified from a whole virus and passaged such that the virus and/or viral RNA replicates and its nucleic acid mutates. Nucleic acid mutations may be point mutations, insertion mutations, or deletion mutations. Nucleic acid mutations may lead to amino acid changes within viral proteins that facilitate growth of the virus in a non-human cell. Adaptation mutations may facilitate phenotypic changes in the virus, including altered plaque size, growth kinetics, temperature sensitivity, drug resistance, virulence, and virus yield in cell culture. These adaptive mutations may be useful in vaccine manufacture by increasing the speed, yield, and consistency of virus cultured in a cell line. Adaptive mutations may change (e.g., enhance or decrease) immunogenicity of viral antigens by altering the structure of immunogenic epitopes. In addition, adaptive mutations may also increase the genetic stability of the virus and/or reduce or otherwise inhibit the development of undesirable mutations in the virus through multiple (e.g., at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more) passages.

Accordingly, in certain embodiments, antigens of the present disclosure include at least one non-human cell adaptation mutation. In certain embodiments, the adaptation mutation is a mutation of a viral antigen to a non-human cell. In some embodiments, the non-human cell is a mammalian cell. Any suitable mammalian cell known in the art may be used, including, without limitation, VERO cells (from monkey kidneys), LLC-MK2 cells (from monkey kidneys), MDBK cells, MDCK cells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219 as described in WO97/37001) cells, BHK21-F cells, HKCC cells, or Chinese hamster ovary cells (CHO cells). In some embodiments, the non-human cell is a monkey cell. In some embodiments, the monkey cell is from a Vero cell line. Any suitable Vero cell line known in the art may be used, including, without limitation, WHO Vero 10-87, ATCC CCL-81, Vero 76 (ATCC Accession No. CRL-1587), or Vero C1008 (ATCC Accession No. CRL-1586). In some embodiments, the Vero cell line is WHO Vero 10-87.

Zika viruses possess a positive sense, single-stranded RNA genome encoding both structural and nonstructural polypeptides. The genome also contains non-coding sequences at both the 5′- and 3′-terminal regions that play a role in virus replication. Structural polypeptides encoded by these viruses include, without limitation, capsid (C), precursor membrane (prM), and envelope (E). Non-structural (NS) polypeptides encoded by these viruses include, without limitation, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

In certain embodiments, antigens of the present disclosure may contain at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, etc.) non-human cell adaptation mutations within one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten) viral antigens/polypeptides, including, without limitation, C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. In some embodiments, antigens of the present disclosure include at least one non-human cell adaptation mutation in Zika virus Non-structural protein 1 (NS1). In some embodiments, antigens of the present disclosure include whole, inactivated virus that may contain at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, etc.) non-human cell adaptation mutations. In some embodiments, antigens of the present disclosure include whole, inactivated virus that may contain at least one non-human cell adaptation mutation in Zika virus Non-structural protein 1 (NS1).

In some embodiments, the at least one non-human cell adaptation mutation is within the NS1 polypeptide. The amino acid sequence of a wild-type, non-cell adapted NS1 polypeptide from an exemplary Zika virus strain is set forth as:

(SEQ ID NO: 1) DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAW EDGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRG PQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAW NSFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGY WIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGP LSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRS TTASGRVIEEWCCRECTMPPLSPRAKDGCWYGMEIRPRKEPESNLVRSMV T. 

In some embodiments, the amino acid sequence of the NS1 polypeptide has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the NS1 polypeptide may be from the amino acid sequence encoded by the sequence of GenBank Accession number KU501215.1 (SEQ ID NO: 2). In some embodiments, the amino acid sequence of the NS1 polypeptide may be amino acid positions 795 to 1145 of the amino acid sequence encoded by the sequence of GenBank Accession number KU501215.1. In some embodiments, the amino acid sequence of the NS1 polypeptide may be from Zika virus strain PRVABC59.

“Sequence Identity”, “% sequence identity”, “% identity”, “% identical” or “sequence alignment” means a comparison of a first amino acid sequence to a second amino acid sequence, or a comparison of a first nucleic acid sequence to a second nucleic acid sequence and is calculated as a percentage based on the comparison. The result of this calculation can be described as “percent identical” or “percent ID.”

Generally, a sequence alignment can be used to calculate the sequence identity by one of two different approaches. In the first approach, both mismatches at a single position and gaps at a single position are counted as non-identical positions in final sequence identity calculation. In the second approach, mismatches at a single position are counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (ignored) as non-identical positions in final sequence identity calculation. In other words, in the second approach gaps are ignored in final sequence identity calculation. The difference between these two approaches, i.e. counting gaps as non-identical positions vs ignoring gaps, at a single position can lead to variability in the sequence identity value between two sequences.

A sequence identity is determined by a program, which produces an alignment, and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in final sequence identity calculation. For example program Needle (EMBOS), which has implemented the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), and which calculates sequence identity per default settings by first producing an alignment between a first sequence and a second sequence, then counting the number of identical positions over the length of the alignment, then dividing the number of identical residues by the length of an alignment, then multiplying this number by 100 to generate the % sequence identity [% sequence identity=(#of Identical residues/length of alignment)×100)].

A sequence identity can be calculated from a pairwise alignment showing both sequences over the full length, so showing the first sequence and the second sequence in their full length (“Global sequence identity”). For example, program Needle (EMBOSS) produces such alignments;% sequence identity=(#of identical residues/length of alignment)×100)].

A sequence identity can be calculated from a pairwise alignment showing only a local region of the first sequence or the second sequence (“Local Identity”). For example, program Blast (NCBI) produces such alignments;% sequence identity=(#of Identical residues/length of alignment)×100)].

The sequence alignment is preferably generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used with the programs default parameter (gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62 for proteins and matrix=EDNAFULL for nucleotides). Then, a sequence identity can be calculated from the alignment showing both sequences over the full length, so showing the first sequence and the second sequence in their full length (“Global sequence identity”). For example:% sequence identity=(#of identical residues/length of alignment)×100)].

In some embodiments, the at least one non-human cell adaptation mutation occurs at one or more amino acid positions within the NS1 polypeptide. In some embodiments, the mutation occurs at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1 using a pairwise alignment algorithm. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution.

In some embodiments, the Zika virus comprises a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1. A position corresponding to position 98 of SEQ ID NO:1 can be determined by aligning the amino acid sequence of an NS-1 protein to SEQ ID NO: 1 using a pairwise alignment algorithm. Amino acid residues in viruses other than Zika virus which correspond to the tryptophan residue at position 98 of SEQ ID NO:1 are shown in FIG. 7 of the present application where these residues are boxed. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution. In some embodiments, the mutation at position 98 is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1.

In some embodiments, antigens of the present disclosure contain at least one non-human cell adaptation mutation within the NS1 protein, and contain at least one mutation (e.g., at least one adaptation mutation) within one or more of the C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 viral proteins. In some embodiments, antigens of the present disclosure contain one or more non-human cell adaptation mutations within the NS1 protein, and do not contain at least one mutation (e.g., at least one non-human cell adaptation mutation) within one or more of the C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 viral proteins. In some embodiments, antigens of the present disclosure contain at least one non-human cell adaptation mutation within the NS1 protein and do not contain at least one mutation (e.g., at least one non-human cell adaptation mutation) within the envelope protein E. In some embodiments, antigens of the present disclosure include whole, inactivated virus that contains at least one non-human cell adaptation mutation in Zika virus Non-structural protein 1 (NS1), and do not include a mutation in Zika virus envelope protein E (Env). In some embodiments, antigens of the present disclosure contain a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 and do not contain any mutation within the envelope protein E. In some embodiments, antigens of the present disclosure include whole, inactivated virus that contains a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 and do not include a mutation in Zika virus envelope protein E (Env). In some embodiments, whole, inactivated virus contains at least one mutation in Zika virus Non-structural protein 1 (NS1) and the sequence encoding the envelope protein is the same as the corresponding sequence in SEQ ID No. 2. In some embodiments, the Zika virus contains a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 and the sequence encoding the envelope protein is the same as the corresponding sequence in SEQ ID No. 2. In some embodiments, whole, inactivated Zika virus contains a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 and the sequence encoding the envelope protein is the same as the corresponding sequence in SEQ ID No. 2.

In some embodiments, antigens of the present disclosure, such as Zika virus, contain at least one non-human cell adaptation mutation that enhances genetic stability as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments, antigens of the present disclosure, such as Zika virus, contain at least one non-human cell adaptation mutation that enhances viral replication as compared to a Zika virus lacking the at least one adaptation mutation. In some embodiments, antigens of the present disclosure, such as Zika virus, contain at least one non-human cell adaptation mutation reduces or otherwise inhibits the occurrence of undesirable mutations, such as within the envelope protein E (Env) of the Zika virus.

In the above embodiments of the present disclosure, an exemplary pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm, using default parameters (e.g. with Gap opening penalty=10.0, and with Gap extension penalty=0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package.

In some embodiments, antigens of the present disclosure from a Zika virus may be used in any of the vaccines and immunogenic compositions of the present disclosure. For example, the antigens of the present disclosure may be useful for treating or preventing Zika virus infection in a human subject in need thereof and/or inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof.

Production of Vaccines and Immunogenic Compositions

Other aspects of the present disclosure relate to Zika virus vaccines and immunogenic compositions containing one or more antigens of the present disclosure from at least one Zika virus. Such vaccines and immunogenic compositions may be useful, for example, for treating or preventing Zika virus infection in a human subject in need thereof and/or inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof. Vaccines and/or immunogenic compositions of the present disclosure may include, without limitation, purified viruses, inactivated viruses, attenuated viruses, recombinant viruses, purified and/or recombinant viral proteins for subunit vaccines. Vaccines and/or immunogenic compositions of the present disclosure may further include a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or a purified inactivated whole virus vaccine or immunogenic composition or an attenuated virus vaccine or immunogenic composition.

Production of vaccines and/or immunogenic compositions of the present disclosure includes growth of Zika virus, with antigens being prepared from the grown virus. Growth in cell culture is a method for preparing vaccines and/or immunogenic compositions of the present disclosure. Cells for viral growth may be cultured in suspension or in adherent conditions.

Cell lines suitable for growth of the at least one virus of the present disclosure are preferably of mammalian origin, and include, but are not limited to: insect cells (e.g., mosquito cells as described herein, VERO cells (from monkey kidneys), horse, cow (e.g. MDBK cells), sheep, dog (e.g. MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO97/37001), cat, and rodent (e.g. hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo. In certain embodiments, the cells are immortalized (e.g. PERC.6 cells, as described in WO 01/38362 and WO 02/40665, and as deposited under ECACC deposit number 96022940). In preferred embodiments, mammalian cells are utilized, and may be selected from and/or derived from one or more of the following non-limiting cell types: fibroblast cells (e.g. dermal, lung), endothelial cells (e.g. aortic, coronary, pulmonary, vascular, dermal microvascular, umbilical), hepatocytes, keratinocytes, immune cells (e.g. T cell, B cell, macrophage, NK, dendritic), mammary cells (e.g. epithelial), smooth muscle cells (e.g. vascular, aortic, coronary, arterial, uterine, bronchial, cervical, retinal pericytes), melanocytes, neural cells (e.g. astrocytes), prostate cells (e.g. epithelial, smooth muscle), renal cells (e.g. epithelial, mesangial, proximal tubule), skeletal cells (e.g. chondrocyte, osteoclast, osteoblast), muscle cells (e.g. myoblast, skeletal, smooth, bronchial), liver cells, retinoblasts, and stromal cells. WO97/37000 and WO97/37001 describe production of animal cells and cell lines that are capable of growth in suspension and in serum free media and are useful in the production and replication of viruses.

Culture conditions for the above cell types are known and described in a variety of publications. Alternatively culture medium, supplements, and conditions may be purchased commercially, such as for example, described in the catalog and additional literature of Cambrex Bioproducts (East Rutherford, N.J.).

In certain embodiments, the cells used in the methods described herein are cultured in serum free and/or protein free media. A medium is referred to as a serum-free medium in the context of the present disclosure in which there are no additives from serum of human or animal origin. Protein-free is understood to mean cultures in which multiplication of the cells occurs with exclusion of proteins, growth factors, other protein additives and non-serum proteins, but can optionally include proteins such as trypsin or other proteases that may be necessary for viral growth. The cells growing in such cultures naturally contain proteins themselves.

Known serum-free media include Iscove's medium, Ultra-CHO medium (BioWhittaker) or EX-CELL (JRH Bioscience). Ordinary serum-containing media include Eagle's Basal Medium (BME) or Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM), which are ordinarily used with up to 10% fetal calf serum or similar additives. Optionally, Minimum Essential Medium (MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified Eagle Medium (DMEM or EDM) may be used without any serum containing supplement. Protein-free media like PF-CHO (JHR Bioscience), chemically-defined media like ProCHO 4CDM (BioWhittaker) or SMIF 7 (Gibco/BRL Life Technologies) and mitogenic peptides like Primactone, Pepticase or HyPep™ (all from Quest International) or lactalbumin hydrolysate (Gibco and other manufacturers) are also adequately known in the prior art. The media additives based on plant hydrolysates have the special advantage that contamination with viruses, mycoplasma or unknown infectious agents can be ruled out.

Cell culture conditions (temperature, cell density, pH value, etc.) are variable over a very wide range owing to the suitability of the cell line employed according to the present disclosure and can be adapted to the requirements of particular viral strains.

The method for propagating virus in cultured cells generally includes the steps of inoculating the cultured cells with the strain to be cultured, cultivating the infected cells for a desired time period for virus propagation, such as for example as determined by virus titer or antigen expression (e.g. between 24 and 168 hours after inoculation) and collecting the propagated virus. In some embodiments, the virus is collected via plaque purification. The cultured cells are inoculated with a virus (measured by PFU or TCID50) to cell ratio of 1:500 to 1:1, preferably 1:100 to 1:5. The virus is added to a suspension of the cells or is applied to a monolayer of the cells, and the virus is absorbed on the cells for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes but usually less than 300 minutes at 25° C. to 40° C., preferably 28° C. to 38° C. The infected cell culture (e.g. monolayers) may be removed either by freeze-thawing or by enzymatic action to increase the viral content of the harvested culture supernatants. The harvested fluids are then either inactivated or stored frozen. Cultured cells may be infected at a multiplicity of infection (“MOI”) of about 0.0001 to 10, preferably 0.002 to 5, more preferably to 0.001 to 2. Still more preferably, the cells are infected at an MOI of about 0.01. Infected cells may be harvested from 30 to 60 hours post infection, or 3 to 10 days post infection. In certain preferred embodiments, the cells are harvested 3 to 7 days post infection. More preferably, the cells are harvested 3 to 5 days post infection. In some embodiments, proteases (e.g., trypsin) may be added during cell culture to allow viral release, and the proteases may be added at any suitable stage during the culture. Alternatively, in certain embodiments, the supernatant of infected cell cultures may be harvested and the virus may be isolated or otherwise purified from the supernatant.

The viral inoculum and the viral culture are preferably free from (i.e. will have been tested for and given a negative result for contamination by) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses, polyomaviruses, birnaviruses, circoviruses, and/or parvoviruses [WO2006/027698].

Where virus has been grown on a cell line then it is standard practice to minimize the amount of residual cell line DNA in the final vaccine, in order to minimize any oncogenic activity of the host cell DNA. Contaminating DNA can be removed during vaccine preparation using standard purification procedures e.g. chromatography, etc. Removal of residual host cell DNA can be enhanced by nuclease treatment e.g. by using a DNase. A convenient method for reducing host cell DNA contamination disclosed in references (Lundblad (2001) Biotechnology and Applied Biochemistry 34:195-197, Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Veterinary Medicine (CVM). May 2001.) involves a two-step treatment, first using a DNase (e.g. Benzonase), which may be used during viral growth, and then a cationic detergent (e.g. CTAB), which may be used during virion disruption. Removal by β-propiolactone treatment can also be used. In one embodiment, the contaminating DNA is removed by benzonase treatment of the culture supernatant.

Production of Antigen

Antigens of the present disclosure for use in vaccines and/or immunogenic compositions including, without limitation, purified viruses, inactivated viruses, inactivated whole viruses, attenuated viruses, recombinant viruses, or purified and/or recombinant viral proteins for subunit vaccines to treat and/or prevent Zika virus infection and/or induce an immune response, such as a protective immune response, against Zika virus, may be produced and/or purified or otherwise isolated by any suitable method known in the art. Antigens of the present disclosure may include, without limitation, whole virus, attenuated virus, inactivated virus, inactivated whole viruses, proteins, polypeptides (including active proteins and individual polypeptide epitopes within proteins), glycopolypeptides, lipopolypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, and carbohydrates produced, derived, purified, and/or otherwise isolated from a Zika virus. For example, suitable antigens may include, without limitation, structural polypeptides such as C, prM, and/or E, and non-structural polypeptides, such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and/or NS5 from Zika virus.

In one embodiment, the antigen of the present disclosure is a purified inactivated whole Zika virus.

Antigen of the present disclosure may be synthesized chemically or enzymatically, produced recombinantly, isolated from a natural source, or a combination of the foregoing. In certain embodiments, antigens of the present disclosure are produced, purified, isolated, and/or derived from at least one Zika virus of the present disclosure. Antigens of the present disclosure may be purified, partially purified, or a crude extract. In some embodiments, antigens of the present disclosure are viruses, such as inactivated viruses, produced as described in the above section entitled “Production of Vaccines and Immunogenic Compositions.”

In certain embodiments, one or more antigens of the present disclosure may be produced by culturing a non-human cell. Cell lines suitable for production of the one or more antigens of the present disclosure may include insect cells (e.g., any of the mosquito cells described herein). Cell lines suitable for production of the one or more antigens of the present disclosure may also be cells of mammalian origin, and include, but are not limited to: VERO cells (from monkey kidneys), horse, cow (e.g. MDBK cells), sheep, dog (e.g. MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 as described in WO97/37001), cat, and rodent (e.g. hamster cells such as BHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), and may be obtained from a wide variety of developmental stages, including for example, adult, neonatal, fetal, and embryo. In certain embodiments, the cells are immortalized (e.g. PERC.6 cells, as described in WO01/38362 and WO02/40665, and as deposited under ECACC deposit number 96022940). In preferred embodiments, mammalian cells are utilized, and may be selected from and/or derived from one or more of the following non-limiting cell types: fibroblast cells (e.g. dermal, lung), endothelial cells (e.g. aortic, coronary, pulmonary, vascular, dermal microvascular, umbilical), hepatocytes, keratinocytes, immune cells (e.g. T cell, B cell, macrophage, NK, dendritic), mammary cells (e.g. epithelial), smooth muscle cells (e.g. vascular, aortic, coronary, arterial, uterine, bronchial, cervical, retinal pericytes), melanocytes, neural cells (e.g. astrocytes), prostate cells (e.g. epithelial, smooth muscle), renal cells (e.g. epithelial, mesangial, proximal tubule), skeletal cells (e.g. chondrocyte, osteoclast, osteoblast), muscle cells (e.g. myoblast, skeletal, smooth, bronchial), liver cells, retinoblasts, and stromal cells. WO97/37000 and WO97/37001 describe production of animal cells and cell lines that capable of growth in suspension and in serum free media and are useful in the production of viral antigens. In certain embodiments, the non-human cell is cultured in serum-free media.

Polypeptide antigens may be isolated from natural sources using standard methods of protein purification known in the art, including, but not limited to, liquid chromatography (e.g., high performance liquid chromatography, fast protein liquid chromatography, etc.), size exclusion chromatography, gel electrophoresis (including one-dimensional gel electrophoresis, two-dimensional gel electrophoresis), affinity chromatography, or other purification technique. In many embodiments, the antigen is a purified antigen, e.g., from about 50% to about 75% pure, from about 75% to about 85% pure, from about 85% to about 90% pure, from about 90% to about 95% pure, from about 95% to about 98% pure, from about 98% to about 99% pure, or greater than 99% pure. The purity of the purified antigen can be determined by size exclusion chromatography and the %-purity corresponds to the % of the main peak to the total area under the curve. The main peak of the purified antigen in the size exclusion chromatography may be more than 85% of the total area under the curve in the size exclusion chromatography, or more than 90% of the total area under the curve in the size exclusion chromatography, or more than 95%, or more than 98% or more than 99% of the total area under the curve in the size exclusion chromatography. Such results are considered as “purified” antigen within the meaning of this invention.

In accordance with the above disclosure regarding purity, the term “purified Zika virus” means that the main peak of the purified Zika virus in the size exclusion chromatography is more than 85% of the total area under the curve in the size exclusion chromatography, or more than 90% of the total area under the curve in the size exclusion chromatography, or more than 95%, more than 98% or more than 99% of the total area under the curve in the size exclusion chromatography.

In accordance with the above disclosure regarding purity, the term “purified inactivated whole Zika virus” means that the main peak of the purified inactivated whole Zika virus in the size exclusion chromatography is more than 85% of the total area under the curve in the size exclusion chromatography, or more than 90% of the total area under the curve in the size exclusion chromatography, or more than 95%, more than 98% or more than 99% of the total area under the curve in the size exclusion chromatography.

One may employ solid phase peptide synthesis techniques, where such techniques are known to those of skill in the art. See Jones, The Chemical Synthesis of Peptides (Clarendon Press, Oxford) (1994). Generally, in such methods a peptide is produced through the sequential addition of activated monomeric units to a solid phase bound growing peptide chain.

Well-established recombinant DNA techniques can be employed for production of polypeptides, where, e.g., an expression construct comprising a nucleotide sequence encoding a polypeptide is introduced into an appropriate host cell (e.g., a eukaryotic host cell grown as a unicellular entity in in vitro cell culture, e.g., a yeast cell, an insect cell, a mammalian cell, etc.) or a prokaryotic cell (e.g., grown in in vitro cell culture), generating a genetically modified host cell; under appropriate culture conditions, the protein is produced by the genetically modified host cell.

Besides killed and attenuated virus immunogenic compositions, one can use a subunit immunogenic composition or other type of immunogenic composition which presents to the animal the antigenic components of Zika virus. The antigenic component may be a protein, glycoprotein, lipid-conjugated protein or glycoprotein, a modified lipid moiety, or other viral component which, when injected into a human, stimulates an immune response in the human such that the human develops protective immunity against Zika virus. For a subunit immunogenic composition, the virus can be cultured on mammalian cells, as described above. The cell culture can be homogenized and an immunogenic composition can be isolated by passage of the cell culture homogenate over the appropriate column or through the appropriate pore size filter or via centrifugation of the cell culture homogenate.

If the antigenic component is a protein, then one can isolate the nucleic acid which encodes that protein and generate an immunogenic composition that contains that isolated nucleic acid. The nucleic acid encoding the antigenic component can be placed on a plasmid downstream of a signal sequence of a eukaryotic promoter. That plasmid can contain one or more selectable markers and be transfected into an attenuated prokaryotic organism, such as Salmonella spp., Shigella spp., or other suitable bacteria. The bacteria can then be administered to the human so that the human can generate a protective immune response to the antigenic component. Alternatively, the nucleic acid encoding the antigenic component can be placed downstream of a prokaryotic promoter, have one or more selectable markers, and be transfected into an attenuated prokaryotic organism such as Salmonella spp., Shigella spp., or other suitable bacteria. The bacteria can then be administered to the eukaryotic human subject for which immune response to the antigen of interest is desired. See, for example, U.S. Pat. No. 6,500,419.

For a subunit immunogenic composition, the nucleic acid encoding a proteinaceous antigenic component of a Zika virus can be cloned into a plasmid such as those described in International Patent Application Publication Number WO 00/32047 (Galen) and International Patent Application Publication Number WO 02/083890 (Galen). Then the plasmid can be transfected into bacteria and the bacteria can produce the desired antigenic protein. One can isolate and purify the desired antigenic protein by a variety of methods described in both patent applications.

Virus Inactivation

Certain aspects of the present disclosure relate to Zika virus vaccines and immunogenic compositions containing one or more antigens from a Zika virus. Vaccines and/or immunogenic compositions of the present disclosure may include a purified virus, a whole virus, a recombinant virus, a live attenuated whole virus or, preferably, an inactivated whole virus, or subunits, polypeptides, and/or antigens from an inactivated virus. As such, certain embodiments of the present disclosure relate to Zika virus vaccines and/or immunogenic compositions containing one or more antigens from at least one inactivated Zika virus.

Methods of inactivating or killing viruses to destroy their ability to infect mammalian cells but do not destroy the structure of the virus are known in the art. Such methods include both chemical and physical means. Suitable means for inactivating a virus include, without limitation, treatment with an effective amount of one or more agents selected from detergents, formalin (also referred to herein as “formaldehyde”), beta-propiolactone (BPL), binary ethylamine (BEI), acetyl ethyleneimine, heat, electromagnetic radiation, x-ray radiation, gamma radiation, ultraviolet radiation (UV radiation), UV-A radiation, UV-B radiation, UV-C radiation, methylene blue, psoralen, carboxyfullerene (C60) and any combination of any thereof. When reference is made herein to a concentration of formaldehyde, it refers to the concentration of formaldehyde (and not to the concentration of formalin). Accordingly, a “formaldehyde concentration of 0.01% (w/v)” refers to 0.01% (w/v) formaldehyde, and no further correction of this concentration for the formaldehyde concentration in the formalin stock solution (which typically contains 37% formaldehyde by mass) has to be made. For example, such a formaldehyde concentration in the virus preparation can be obtained by diluting formalin to a working solution having a formaldehyde content of 1.85% (w/v) which is then further diluted to the required concentration when it is mixed with the virus preparation such as the Zika virus preparation.

In certain embodiments of the present disclosure the at least one virus is chemically inactivated. Agents for chemical inactivation and methods of chemical inactivation are well-known in the art and described herein. In some embodiments, the at least one virus is chemically inactivated with one or more of BPL, formalin, or BEI. In certain embodiments where the at least one virus is chemically inactivated with BPL, the virus may contain one or more modifications. In some embodiments, the one or more modifications may include a modified nucleic acid. In some embodiments, the modified nucleic acid is an alkylated nucleic acid. In other embodiments, the one or more modifications may include a modified polypeptide. In some embodiments, the modified polypeptide contains a modified amino acid residue including one or more of a modified cysteine, methionine, histidine, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine.

In certain embodiments where the at least one virus is chemically inactivated with formalin, the inactivated virus may contain one or more modifications. In some embodiments, the one or more modifications may include a modified polypeptide. In some embodiments, the one or more modifications may include a cross-linked polypeptide. In some embodiments where the at least one virus is chemically inactivated with formalin, the vaccine or immunogenic composition further includes formalin. In certain embodiments where the at least one virus is chemically inactivated with BEI, the virus may contain one or more modifications. In some embodiments, the one or more modifications may include a modified nucleic acid. In some embodiments, the modified nucleic acid is an alkylated nucleic acid.

In some embodiments where the at least one virus is chemically inactivated with formalin, any residual unreacted formalin may be neutralized with sodium metabisulfite, may be dialyzed out, and/or may be buffer exchanged to remove the residual unreacted formalin. In some embodiments, the sodium metabisulfite is added in excess. In some embodiments, the solutions may be mixed using a mixer, such as an in-line static mixer, and subsequently filtered or further purified (e.g., using a cross flow filtrations system).

Certain embodiments of the present disclosure relate to a method for inactivating a Zika virus preparation. In some embodiments, the method involves (a) isolating, followed by purification the Zika virus preparation from one or more non-human cells that are used to produce the virus preparation and (b) treating the virus preparation with an effective amount of formalin.

Certain embodiments of the present disclosure relate to a method for inactivating a Zika virus preparation. In some embodiments, the method comprises:

(a) isolating the Zika virus preparation from one or more cells cultured in vitro, wherein the cells are used to produce the Zika virus preparation, wherein isolating the Zika virus preparation comprises one or more steps selected from: (i) depth filtration, (ii) buffer exchange and/or dilution; (iii) ion exchange chromatography; and (b) treating the Zika virus preparation with formaldehyde, wherein the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.025 to 0.5.

In certain embodiments, treating with an effective amount of formalin includes, without limitation, treating with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v. For example, treating with an effective amount of formalin may include treating with formalin in an amount that ranges from about 0.001% to about 3.0% v/v, about 0.005% to about 2.0% v/v, or about 0.01% to about 1.0% v/v, or in an amount of about 0.001%, about 0.0025%, about 0.005%, about 0.0075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.25%, about 1.5%, about 1.75%, about 2.0%, about 2.25%, about 2.5%, about 2.75%, or about 3.0% v/v.

In certain embodiments of the method, the Zika virus preparation is treated with formalin at a temperature that ranges from about 2° C. to about 42° C. For example, the Zika virus preparation may be treated with formalin at a temperature that ranges from about 2° C. to about 42° C., about 2° C. to about 8° C., about 15° C. to about 37° C. about 17° C. to about 27° C., about 20° C. to about 25° C., or at a temperature of about 2° C., about 4° C., about 8° C., about 10° C., about 15° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 37° C., or about 42° C. In some embodiments, the Zika virus preparation is treated with formalin at room temperature.

In some embodiments, the Zika virus preparation is treated with formalin for at least about 1 day. For example, the Zika virus preparation may be treated with formalin for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, and e.g. for no more than 15 days, e.g. from 5 to 15 days. For example, the Zika virus preparation may be treated with formalin for at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 21 days, at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days, at least about 29 days, at least about 30 days, or more. In some embodiments, the Zika virus preparation is treated with formalin for at least about 9 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 11 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 14 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 20 days. In some embodiments, the Zika virus preparation is treated with formalin for at least about 30 days.

In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for eight to twelve days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for nine to eleven days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for eight to twelve days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for nine to eleven days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for ten days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for eight to twelve days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for nine to eleven days at a temperature of 15° C. to 30° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for ten days at a temperature of 15° C. to 30° C.

In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for eight to twelve days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for nine to eleven days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for eight to twelve days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for nine to eleven days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for ten days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for eight to twelve days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for nine to eleven days at a temperature of 18° C. to 25° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for ten days at a temperature of 18° C. to 25° C.

In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for eight to twelve days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for nine to eleven days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.005 to 0.02% (w/v) formalin for ten days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for eight to twelve days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for nine to eleven days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.008 to 0.015% (w/v) formalin for ten days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for eight to twelve days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for nine to eleven days at a temperature of 22° C. In some embodiments, the Zika virus preparation is treated with 0.01% (w/v) formalin for ten days at a temperature of 22° C.

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.05 to 0.25. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.075 to 0.15. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.1.

In some embodiments, the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the formaldehyde concentration is 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the formaldehyde concentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.025 to 0.5 and the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.025 to 0.5 and the formaldehyde concentration is 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.025 to 0.5 and the formaldehyde concentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.05 to 0.25 and the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.05 to 0.25 and the formaldehyde concentration is 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.05 to 0.25 and the formaldehyde concentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.075 to 0.15 and the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.075 to 0.15 and the formaldehyde concentration is 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.075 to 0.15 and the formaldehyde concentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.1 and the formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.1 and the formaldehyde concentration is 0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.1 and the formaldehyde concentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.025 to 0.5 and the period of incubation with formaldehyde is eight to twelve days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.025 to 0.5 and the period of incubation with formaldehyde is nine to eleven days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.025 to 0.5 and the period of incubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.05 to 0.25 and the period of incubation with formaldehyde is eight to twelve days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.05 to 0.25 and the period of incubation with formaldehyde is nine to eleven days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.05 to 0.25 and the period of incubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.075 to 0.15 and the period of incubation with formaldehyde is eight to twelve days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.075 to 0.15 and the period of incubation with formaldehyde is nine to eleven days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.075 to 0.15 and the period of incubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of the formaldehyde concentration as measured in % (w/v) with the period of incubation with formaldehyde as measured in days is 0.1 and the period of incubation with formaldehyde is eight to twelve days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.1 and the period of incubation with formaldehyde is nine to eleven days. In some embodiments, the numerical result of the multiplication of the formaldehyde concentration with the period of incubation with formaldehyde is 0.1 and the period of incubation with formaldehyde is ten days.

In some embodiments, the method further involves neutralizing unreacted formalin with an effective amount of sodium metabisulfite. In some embodiments, the effective amount of sodium metabisulfite ranges from about 0.01 mM to about 100 mM. For example, the sodium metabisulfite may be added at an effective concentration of from about 0.01 mM to about 100 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 20 mM, or from about 1 mM to about 10 mM, or at a concentration of about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM about 40 mM, about 50 mM, about 75 mM or about 100 mM. In some embodiments, the formalin is neutralized with about 2 mM sodium metabisulfite.

In some embodiments, the method involves (a) isolating followed by purification of the Zika virus preparation from one or more non-human cells that are used to produce the virus preparation; (b) treating the virus preparation with an effective amount of formalin; (c) neutralizing the virus preparation with an effective amount of sodium metabisulfite; and (d) purifying the neutralized virus preparation. Any method of purifying a virus preparation known in the art may be employed, including, without limitation, using cross flow filtration (CFF), multimodal chromatography, size exclusion chromatography, cation exchange chromatography, and/or anion exchange chromatography. In some embodiments, the neutralized virus preparation is purified by cross flow filtration (CFF). In some embodiments, the virus preparation is purified to a high degree in an amount that is about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95% about 96%, about 97%, about 98%, about 99%, or more.

Certain embodiments of the present disclosure relate thus to Zika virus vaccines and/or immunogenic compositions containing a purified inactivated whole Zika virus. The term “inactivated whole Zika virus” as used herein is intended to comprise a Zika virus, which has been treated with an inactivating method such as treatment with an effective amount of formalin. Such a treatment is considered not to destroy the structure of the virus, i.e. it does not destroy the secondary, tertiary or quaternary structure and immunogenic epitopes of the virus, but the inactivated Zika virus is no longer able to infect host cells, which can be infected with a Zika virus that has not been inactivated. In one embodiment, the inactivated Zika virus is no longer able to infect VERO cells and exert a cytopathic effect on the VERO cells.

The method for determining the completeness of inactivation of an Zika virus preparation comprises the steps of:

-   -   (i) inoculating insect cells with an Zika virus preparation         which was subjected to an inactivation step and incubating the         insect cells for a first period of time, thereby producing an         insect cell supernatant;     -   (ii) inoculating mammalian cells with the insect cell         supernatant produced in (i) and incubating the mammalian cells         for a second period of time; and     -   (iii) determining whether the virus preparation contains a         residual replicating virus that produces a cytopathic effect on         the mammalian cells.

In some embodiments, the method for determining the completeness of inactivation of an Zika virus preparation comprises the following steps:

-   -   (i) inoculating C6/36 cells with a Zika virus preparation which         was subjected to an inactivation step and incubating the C6/36         cells for a first period of time, thereby producing an C6/36         cell supernatant;     -   (ii) inoculating Vero cells with the C6/36 cell supernatant         produced in (i) and incubating the Vero cells for a second         period of time; and     -   (iii) determining whether the virus preparation contains a         residual replicating virus that produces a cytopathic effect on         the Vero cells.

At the end of the second period of time it is determined whether the virus preparation has a cytopathic effect on the mammalian cells. A cytopathic effect is any change in the cell structure caused by viral invasion, infection, and budding from the cells during viral replication. In the method of the present disclosure, the cytopathic effect is determined by a change in the media color from pink to orange or yellow, if the cells are cultured in a medium containing phenol red, or by a microscopic examination of the mammalian cells. If the microscopic examination of the mammalian cells shows that the cells round, begin to pull away from the tissue culture vessel (plate, well or flask), or clear from the tissue culture plate/flask, it is considered that a cytopathic effect is present. Other indicia of a cytopathic effect include the fusion of adjacent cells to form syncytia and the appearance of nuclear or cytoplasmic inclusion bodies.

As discussed above, the method disclosed herein has a very low limit of detection. With this method a virus content of less than 1.0 TCID50 can be detected. In some embodiments, a virus content of less than 0.8 TCID50 can be detected. In some embodiments, a virus content of less than 0.5 TCID50 can be detected. In some embodiments, a virus content of less than 0.2 TCID50 can be detected. In some embodiments, a virus content of less than 0.1 TCID50 can be detected.

For the present disclosure the term “inactivated whole Zika virus” thus refers in particular to a Zika virus obtainable from a method wherein the Zika virus is treated with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperature that ranges from about 15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C. The definition is meant to encompass Zika virus obtained from a method wherein the Zika virus is treated with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperature that ranges from about 15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C., but is not to be understood to be limited to those, since other methods may lead to the same inactivated whole Zika virus. In certain such embodiments, however, the Zika virus is obtained from a method wherein the Zika virus is treated with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperature that ranges from about 15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C.

Alternatively, within the present disclosure “inactivated whole Zika virus” thus refers to a Zika virus that has been tested by the method comprising the steps (i) to (iii) and does not show any plaque formation in step (iii):

-   -   (i) inoculating insect cells with an Zika virus preparation         which was subjected to an inactivation step and incubating the         insect cells for a first period of time, thereby producing an         insect cell supernatant;     -   (ii) inoculating mammalian cells with the insect cell         supernatant produced in (i) and incubating the mammalian cells         for a second period of time; and     -   (iii) determining whether the virus preparation contains a         residual replicating virus that produces a cytopathic effect on         the mammalian cells.

The term “purified inactivated whole Zika virus” thus refers to a Zika virus obtainable or obtained from a method wherein the Zika virus is treated with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperature that ranges from about 15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C., or alternatively by the above mentioned method for determining the completeness of inactivation and, if required, has been subjected to a purification process. The purified Zika virus has therefore a lower content of host cell proteins such as Vero cell proteins and host cell DNA such as Vero cell DNA than a non-purified Zika virus. The term “purified inactivated whole Zika virus” thus refers to a Zika virus obtainable or obtained from a method wherein the Zika virus is treated with formalin in an amount that ranges from about 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperature that ranges from about 15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C., or alternatively by the above mentioned method for determining the completeness of inactivation and, provides a main peak of at least 85% of the total area under the curve in the size exclusion chromatography.

In certain such embodiments the purified inactivated whole Zika virus is furthermore a clonal isolate obtained or obtainable by plaque purification.

In certain such embodiments the purified inactivated whole Zika virus, which is optionally furthermore a clonal isolate obtained or obtainable by plaque purification, contains a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO: 1 and does not contain any mutation within the envelope protein E. In certain such embodiments the mutation is a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1. In certain such embodiments the Zika virus is derived from strain PRVABC59. In certain such embodiments the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2.

The vaccines and/or immunogenic compositions of the present disclosure containing one or more antigens from at least one inactivated Zika virus may be useful for treating or preventing Zika virus infection in a human subject in need thereof and/or inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof.

Other aspects of the present disclosure relate to vaccine or immunogenic composition comprising an inactivated Zika virus which is obtainable by the methods disclosed herein. These vaccines or immunogenic compositions have a particularly low content of residual formaldehyde.

The term “residual formaldehyde content” refers to the amount of formaldehyde which is still present in the vaccine or immunogenic composition after the Zika virus has been inactivated and the preparation has been neutralized and optionally subjected to one or more further purification or filtration steps. According to the US pharmacopoeia the upper limit for residual formaldehyde in vaccines comprising inactivated bacteria or viruses is 0.02% which is equivalent to 100 μg/ml formaldehyde.

The residual formaldehyde content can be determined by any method known to the skilled person. One suitable method is described in EMEA, VICH Topic GL25, Biologicals: Testing of residual formaldehyde, 30 Apr. 2002 and involves the use of Methylbenzothiazolone hydrazone hydrochloride (MBTH). Other methods include acetyl acetone titration, ferric chloride titration and the basic fuchsin test. A particularly suitable method is described herein.

The vaccine or immunogenic composition is in a form which can be administered to a subject and typically contains one or more pharmaceutically acceptable excipients.

The content of residual formaldehyde in the vaccine or immunogenic composition is less than 50 μg/ml. In one embodiment, the residual formaldehyde content in the vaccine or immunogenic composition is less than 45 μg/ml, less than 40 μg/ml, less than 35 μg/ml, less than 30 μg/ml, less than 25 μg/ml, less than 20 μg/ml, less than 15 μg/ml or less than 10 μg/ml. In one embodiment, the residual formaldehyde content in the vaccine or immunogenic composition is less than 9.5 μg/ml, less than 9 μg/ml, less than 8.5 μg/ml, less than 8 μg/ml, less than 7.5 μg/ml, less than 7 μg/ml, less than 6.5 μg/ml, less than 6 μg/ml, less than 5.5 μg/ml, less than 5 μg/ml, less than 4.5 μg/ml, less than 4 μg/ml, less than 3.5 μg/ml, less than 3 μg/ml, less than 2.5 μg/ml, less than 2 μg/ml, less than 1.5 μg/ml, less than 1 μg/ml or less than 0.5 μg/ml. In one embodiment, the residual formaldehyde content in the vaccine or immunogenic composition is less than 0.5 μg/ml.

Methods for Determining Residual Formaldehyde Content

Other aspects of the present disclosure relate to a method for determining the residual formaldehyde content in a vaccine or immunogenic composition comprising an inactivated virus, comprising the steps of:

(a) providing a composition comprising a virus which has been treated with formaldehyde; (b) mixing the composition of (a) with phosphoric acid and 2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture; (c) incubating the mixture of (b) under suitable conditions; and (d) analyzing the mixture for the presence of residual formaldehyde.

The use of DNPH as detection reagent offers the following advantages: (1) high sensitivity, (2) UV detection of the derivatized formaldehyde and (3) one-step sample preparation without heating. The present method is particularly suitable for detecting residual formaldehyde in vaccines containing an adjuvant such as aluminum hydroxide. The method was validated in terms of specificity, linearity, accuracy, repeatability, robustness and stability according to the International Conference on Harmonization (ICH) Q2 guidelines. In some embodiments 50 parts of the composition comprising a virus which has been treated with formaldehyde are mixed with 1 part of 15 to 25% (v/v) phosphoric acid and 2.5 parts of 0.9 to 1.1 mg/ml DNPH. In some embodiments 50 parts of the composition comprising a virus which has been treated with formaldehyde are mixed with 1 part of 20% (v/v) phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH.

In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 18° C. to 30° C. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 20° C. to 25° C. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 22° C.

In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated for 10 to 30 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated for 15 to 25 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated for 20 minutes.

In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 18° C. to 30° C. for 10 to 30 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 18° C. to 30° C. for 15 to 25 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 18° C. to 30° C. for 20 minutes.

In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 20° C. to 25° C. for 10 to 30 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 20° C. to 25° C. for 15 to 25 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 20° C. to 25° C. for 20 minutes.

In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 22° C. for 10 to 30 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 22° C. for 15 to 25 minutes. In some embodiments the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is incubated at a temperature of 22° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 18° C. to 30° C. for 10 to 30 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 18° C. to 30° C. for 15 to 25 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 18° C. to 30° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 20° C. to 25° C. for 10 to 30 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 20° C. to 25° C. for 15 to 25 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 20° C. to 25° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 22° C. for 10 to 30 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 22° C. for 15 to 25 minutes. In some embodiments the mixture of 50 parts of the composition comprising a virus which has been treated with formaldehyde with 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at a temperature of 22° C. for 20 minutes.

After incubation, the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH may be analyzed by any suitable method. In one embodiment, after incubation, the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is analyzed by HPLC. In one embodiment, after incubation, the mixture of the composition comprising a virus which has been treated with formaldehyde with phosphoric acid and DNPH is analyzed by reverse phase HPLC. In one embodiment, the ligand of the reversed phase HPLC column is selected from C18, n-butal, n-octyl, phenyl and cyanopropyl. In one embodiment, the ligand of the reversed phase HPLC column is C18. In one embodiment, a mixture of water and acetonitrile (1:1, v/v) is used as the mobile phase in the reversed phase HPLC. In one embodiment, the detection wavelength is 360 nm.

In one embodiment, the present disclosure provides a method for determining the residual formaldehyde content in a vaccine or immunogenic composition comprising an inactivated virus, comprising the steps of:

-   -   (a) providing a composition comprising a virus which has been         treated with formaldehyde;     -   (b) mixing 50 parts of the composition of (a) with 1 part of 20%         phosphoric acid and 2.5 parts of 1 mg/ml         2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;     -   (c) incubating the mixture of (b) for 20 minutes at room         temperature; and     -   (d) analyzing the mixture for the presence of residual         formaldehyde by reversed phase HPLC using a C18 column and a         mixture of water and acetonitrile (1:1, v/v) as the mobile         phase.

In one embodiment, the present disclosure provides a method for determining the residual formaldehyde content in a vaccine or immunogenic composition comprising an inactivated Zika virus, comprising the steps of:

-   -   (a) providing a composition comprising a Zika virus which has         been treated with formaldehyde;     -   (b) mixing 50 parts of the composition of (a) with 1 part of 20%         phosphoric acid and 2.5 parts of 1 mg/ml         2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;     -   (c) incubating the mixture of (b) for 20 minutes at room         temperature; and     -   (d) analyzing the mixture for the presence of residual         formaldehyde by reversed phase HPLC using a C18 column and a         mixture of water and acetonitrile (1:1, v/v) as the mobile         phase.

Adjuvants

Other aspects of the present disclosure relate to Zika virus vaccines and/or immunogenic compositions containing one or more antigens from at least one Zika virus described herein in combination with one or more adjuvants. Such adjuvanted vaccines and/or immunogenic compositions of the present disclosure may be useful for treating or preventing Zika virus infection in a human subject in need thereof and/or inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof.

Various methods of achieving an adjuvant effect for vaccines are known and may be used in conjunction with the Zika virus vaccines and/or immunogenic compositions disclosed herein. General principles and methods are detailed in “The Theory and Practical Application of Adjuvants”, 1995, Duncan E. S. Stewart-Tull (ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines: New Generation Immunological Adjuvants”, 1995, Gregoriadis G et al. (eds.), Plenum Press, New York, ISBN 0-306-45283-9.

In some embodiments, a Zika virus vaccine or immunogenic composition includes the antigens and an adjuvant. Antigens may be in a mixture with at least one adjuvant, at a weight-based ratio of from about 10:1 to about 10¹⁰:1 antigen:adjuvant, e.g., from about 10:1 to about 100:1, from about 100:1 to about 10³:1, from about 10³:1 to about 10⁴:1, from about 10⁴:1 to about 10⁵:1, from about 10⁵:1 to about 10⁶:1, from about 10⁶:1 to about 10⁷:1, from about 10⁷:1 to about 10⁸:1, from about 10⁸:1 to about 10⁹:1, or from about 10⁹:1 to about 10¹⁰:1 antigen:adjuvant. One of skill in the art can readily determine the appropriate ratio through information regarding the adjuvant and routine experimentation to determine optimal ratios.

Exemplary adjuvants may include, but are not limited to, aluminum salts, calcium phosphate, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA), MLA derivatives, synthetic lipid A, lipid A mimetics or analogs, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions (oil emulsions), chitosan, vitamin D, stearyl or octadecyl tyrosine, virosomes, cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, liposomes, Complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant is an aluminum salt.

In some embodiments, the adjuvant includes at least one of alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, aluminum salt adjuvants of the present disclosure have been found to increase adsorption of the antigens of the Zika virus vaccines and/or immunogenic compositions of the present disclosure. Accordingly, in some embodiments, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the antigen is adsorbed to the aluminum salt adjuvant.

In some embodiments, the vaccine or immunogenic composition includes an aluminum salt adjuvant (e.g., alum) from about 100 μg to about 600 μg, from about 100 μg to about 500 μg, from about 125 μg to about 500 μg, from about 150 μg to about 500 μg, from about 175 μg to about 500 μg, from about 100 μg to about 450 μg, from about 125 μg to about 450 μg, from about 150 μg to about 450 μg, from about 175 μg to about 450 μg, from about 100 μg to about 400 μg, from about 125 μg to about 400 μg, from about 150 μg to about 400 μg, from about 175 μg to about 400 μg, from about 100 μg to about 350 μg, from about 125 μg to about 350 μg, from about 150 μg to about 350 μg, from about 175 μg to about 350 μg, from about 100 μg to about 300 μg, from about 125 μg to about 300 μg, from about 150 μg to about 300 μg, from about 175 μg to about 300 μg, from about 100 μg to about 250 μg, from about 125 μg to about 250 μg, from about 150 μg to about 250 μg, from about 175 μg to about 250 μg, from about 100 μg to about 225 μg, from about 125 μg to about 225 μg, from about 150 μg to about 225 μg, from about 175 μg to about 225 μg, or about 200 μg. In some embodiments the vaccine or immunogenic composition includes an aluminum salt adjuvant (e.g., alum such as aluminum hydroxide) at about 100 μg to about 600 μg at about 100 μg to about 300 μg or about 150 μg to about 250 μg or about 200 μg.

In some embodiments, the vaccines and/or immunogenic compositions contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1 as described herein in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole plaque purified Zika virus isolate comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

Certain embodiments of the present disclosure include a method for preparing an adjuvanted Zika virus vaccine or immunogenic composition, which involves (a) mixing the vaccine or immunogenic composition with an aluminum salt adjuvant, with the vaccine or immunogenic composition including one or more antigens from at least one Zika virus described herein and (b) incubating the mixture under suitable conditions for a period of time that ranges from about 1 hour to about 24 hours (e.g., about 16 hours to about 24 hours), with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% of the antigen adsorbed to the aluminum salt adjuvant. In certain embodiments of the method, the at least one Zika virus is a Zika virus comprising a non-human cell adaptation mutation (e.g., a non-human cell adaptation mutation in protein NS1 such as a Trp98Gly mutation.

In some embodiments of the method, the mixture is incubated at a temperature that ranges from about 2° C. to about 8° C. In some embodiments of the method, the mixture is incubated under constant mixing using any suitable mixer known in the art. In some embodiments of the method, the mixture is incubated at pH that ranges in value from about 6.5 to about 8.5, from about 6.5 to about 8, from about 6.8 to about 7.8, from about 6.9 to about 7.6, from about 7 to about 7.5, from about 6.8 to about 8.5, from about 6.9 to about 8.5, or from about 7 to about 8.5. In certain preferred embodiments, the mixture is incubated at a neutral pH. In some embodiments of the method, the aluminum salt adjuvant is selected from alum, aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, and Alhydrogel 85.

Monophosphoryl Lipid A (MLA), a non-toxic derivative of lipid A from Salmonella, is a potent TLR-4 agonist that has been developed as a vaccine adjuvant (Evans et al. (2003) Expert Rev. Vaccines 2(2): 219-229). In pre-clinical murine studies intranasal MLA has been shown to enhance secretory, as well as systemic, humoral responses (Baldridge et al. (2000) Vaccine 18(22): 2416-2425; Yang et al. (2002) Infect. Immun 70(7): 3557-3565). It has also been proven to be safe and effective as a vaccine adjuvant in clinical studies of greater than 120,000 patients (Baldrick et al. (2002) Regul. Toxicol. Pharmacol. 35(3): 398-413; Baldrick et al. (2004) J. Appl. Toxicol. 24(4): 261-268). MLA stimulates the induction of innate immunity through the TLR-4 receptor and is thus capable of eliciting nonspecific immune responses against a wide range of infectious pathogens, including both gram negative and gram positive bacteria, viruses, and parasites (Baldrick et al. (2004) J. Appl. Toxicol. 24(4): 261-268; Persing et al. (2002) Trends Microbiol. 10(10 Suppl): S32-37). Inclusion of MLA in intranasal formulations should provide rapid induction of innate responses, eliciting nonspecific immune responses from viral challenge while enhancing the specific responses generated by the antigenic components of the vaccine.

Accordingly, in one embodiment, the present disclosure provides a composition comprising monophosphoryl lipid A (MLA), 3 De-O-acylated monophosphoryl lipid A (3D-MLA), or a derivative thereof as an enhancer of adaptive and innate immunity. Chemically 3D-MLA is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA). In another embodiment, the present disclosure provides a composition comprising synthetic lipid A, lipid A mimetics or analogs, such as BioMira's PET Lipid A, or synthetic derivatives designed to function like TLR-4 agonists.

Additional exemplary adjuvants include, without limitation, polypeptide adjuvants that may be readily added to the antigens described herein by co-expression with the polypeptide components or fusion with the polypeptide components to produce chimeric polypeptides. Bacterial flagellin, the major protein constituent of flagella, is an adjuvant which has received increasing attention as an adjuvant protein because of its recognition by the innate immune system by the toll-like receptor TLR5. Flagellin signaling through TLR5 has effects on both innate and adaptive immune functions by inducing DC maturation and migration as well as activation of macrophages, neutrophils, and intestinal epithelial cells resulting in production of pro-inflammatory mediators.

TLR5 recognizes a conserved structure within flagellin monomers that is unique to this protein and is required for flagellar function, precluding its mutation in response to immunological pressure. The receptor is sensitive to a 100 fM concentration but does not recognize intact filaments. Flagellar disassembly into monomers is required for binding and stimulation.

As an adjuvant, flagellin has potent activity for induction of protective responses for heterologous antigens administered either parenterally or intranasally and adjuvant effects for DNA vaccines have also been reported. A Th2 bias is observed when flagellin is employed which would be appropriate for a respiratory virus such as influenza but no evidence for IgE induction in mice or monkeys has been observed. In addition, no local or systemic inflammatory responses have been reported following intranasal or systemic administration in monkeys. The Th2 character of responses elicited following use of flagellin is somewhat surprising since flagellin signals through TLR5 in a MyD88-dependent manner and all other MyD88-dependent signals through TLRs have been shown to result in a Th1 bias. Importantly, pre-existing antibodies to flagellin have no appreciable effect on adjuvant efficacy making it attractive as a multi-use adjuvant.

A common theme in many recent intranasal vaccine trials is the use of adjuvants and/or delivery systems to improve vaccine efficacy. In one such study an influenza H3 vaccine containing a genetically detoxified E. coli heat-labile enterotoxin adjuvant (LT R192G) resulted in heterosubtypic protection against H5 challenge but only following intranasal delivery. Protection was based on the induction of cross neutralizing antibodies and demonstrated important implications for the intranasal route in development of new vaccines.

Cytokines, colony-stimulating factors (e.g., GM-CSF, CSF, and the like); tumor necrosis factor; interleukin-2, -7, -12, interferons and other like growth factors, may also be used as adjuvants as they may be readily included in the Zika virus vaccines or immunogenic compositions by admixing or fusion with the polypeptide component.

In some embodiments, the Zika virus vaccine and/or immunogenic compositions disclosed herein may include other adjuvants that act through a Toll-like receptor such as a nucleic acid TLR9 ligand comprising a 5′-TCG-3′ sequence; an imidazoquinoline TLR7 ligand; a substituted guanine TLR7/8 ligand; other TLR7 ligands such as Loxoribine, 7-deazadeoxyguanosine, 7-thia-8-oxodeoxyguanosine, Imiquimod (R-837), and Resiquimod (R-848).

Certain adjuvants facilitate uptake of the vaccine molecules by APCs, such as dendritic cells, and activate these. Non-limiting examples are selected from the group consisting of an immune targeting adjuvant; an immune modulating adjuvant such as a toxin, a cytokine, and a mycobacterial derivative; an oil formulation; a polymer; a micelle forming adjuvant; a saponin; an immunostimulating complex matrix (ISCOM matrix); a particle; DDA; aluminum adjuvants; DNA adjuvants; MLA; and an encapsulating adjuvant.

Additional examples of adjuvants include agents such as aluminum salts such as hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in buffered saline (see, e.g., Nicklas (1992) Res. Immunol. 143:489-493), admixture with synthetic polymers of sugars (e.g. Carbopol®) used as 0.25 percent solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° C. to 101° C. for 30 second to 2 minute periods respectively and also aggregation by means of cross-linking agents are possible. Aggregation by reactivation with pepsin treated antibodies (Fab fragments) to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Admixture with oils such as squalene and IFA may also be used.

DDA (dimethyldioctadecylammonium bromide) is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as quillaja saponins such as QuilA and QS21 are interesting. Further possibilities include poly[di(earboxylatophenoxy)phosphazene (PCPP) derivatives of lipopolysaccharides such as monophosphoryl lipid A (MLA), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP). The lipopolysaccharide based adjuvants may also be used for producing a predominantly Th1-type response including, for example, a combination of monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt.

Liposome formulations are also known to confer adjuvant effects, and therefore liposome adjuvants may be used in conjunction with the Zika virus vaccines and/or immunogenic compositions.

Immunostimulating complex matrix type (ISCOM® matrix) adjuvants may also be used with the Zika virus vaccine antigens and immunogenic compositions, especially since it has been shown that this type of adjuvants are capable of up-regulating MHC Class II expression by APCs. An ISCOM matrix consists of (optionally fractionated) saponins (triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid. When admixed with the immunogenic protein such as the Zika virus vaccine or immunogenic composition antigens, the resulting particulate formulation is what is known as an ISCOM particle where the saponin may constitute 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, and the protein 10-15% w/w. Details relating to composition and use of immunostimulating complexes can for example be found in the above-mentioned text-books dealing with adjuvants, but also Morein B et al. (1995) Clin. Immunother. 3: 461-475 as well as Barr I G and Mitchell G F (1996) Immunol. and Cell Biol. 74: 8-25 provide useful instructions for the preparation of complete immunostimulating complexes.

The saponins, whether or not in the form of iscoms, that may be used in the adjuvant combinations with the Zika virus vaccines and immunogenic compositions disclosed herein include those derived from the bark of Quillaja Saponaria Molina, termed Quil A, and fractions thereof, described in U.S. Pat. No. 5,057,540 and “Saponins as vaccine adjuvants”, Kensil, C. R. (1996) Crit Rev Ther Drug Carrier Syst 12 (1-2):1-55; and EP 0 362 279 B1. Exemplary fractions of Quil A are QS21, QS7, and QS17.

β-Escin is another hemolytic saponins for use in the adjuvant compositions of the Zika virus vaccines and/or immunogenic compositions. Escin is described in the Merck index (12th ed: entry 3737) as a mixture of saponins occurring in the seed of the horse chestnut tree, Lat: Aesculus hippocastanum. Its isolation is described by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and by ion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractions of escin have been purified and shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August; 44(8):1454-1464)). β-escin is also known as aescin.

Another hemolytic saponin for use in the Zika virus vaccines and/or immunogenic compositions is Digitonin. Digitonin is described in the Merck index (12th Edition, entry 3204) as a saponin, being derived from the seeds of Digitalis purpurea and purified according to the procedure described Gisvold et al. (1934) J. Am. Pharm. Assoc. 23: 664; and Ruhenstroth-Bauer (1955) Physiol. Chem., 301, 621. Its use is described as being a clinical reagent for cholesterol determination.

Another interesting possibility of achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992. In brief, the presentation of a relevant antigen such as an antigen in a Zika virus vaccine and/or immunogenic composition of the present disclosure can be enhanced by conjugating the antigen to antibodies (or antigen binding antibody fragments) against the FC receptors on monocytes/macrophages. Especially conjugates between antigen and anti-FCRI have been demonstrated to enhance immunogenicity for the purposes of vaccination. The antibody may be conjugated to the Zika virus vaccine or immunogenic composition antigens after generation or as a part of the generation including by expressing as a fusion to any one of the polypeptide components of the Zika virus vaccine and/or immunogenic composition antigens. Other possibilities involve the use of the targeting and immune modulating substances (e.g., cytokines). In addition, synthetic inducers of cytokines such as poly I:C may also be used.

Suitable mycobacterial derivatives may be selected from the group consisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, (Ribi ImmunoChem Research Inc., Hamilton, Mont.) and a diester of trehalose such as TDM and TDE.

Examples of suitable immune targeting adjuvants include CD40 ligand and CD40 antibodies or specifically binding fragments thereof (cf. the discussion above), mannose, a Fab fragment, and CTLA-4.

Examples of suitable polymer adjuvants include a carbohydrate such as dextran, PEG, starch, mannan, and mannose; a plastic polymer; and latex such as latex beads.

Yet another interesting way of modulating an immune response is to include the immunogen (optionally together with adjuvants and pharmaceutically acceptable carriers and vehicles) in a “virtual lymph node” (VLN) (a proprietary medical device developed by ImmunoTherapy, Inc., 360 Lexington Avenue, New York, N.Y. 10017-6501). The VLN (a thin tubular device) mimics the structure and function of a lymph node. Insertion of a VLN under the skin creates a site of sterile inflammation with an upsurge of cytokines and chemokines. T- and B-cells as well as APCs rapidly respond to the danger signals, home to the inflamed site and accumulate inside the porous matrix of the VLN. It has been shown that the necessary antigen dose required to mount an immune response to an antigen is reduced when using the VLN, and that immune protection conferred by vaccination using a VLN surpassed conventional immunization using Ribi as an adjuvant. The technology is described briefly in Gelber C et al., 1998, “Elicitation of Robust Cellular and Humoral Immune Responses to Small Amounts of Immunogens Using a Novel Medical Device Designated the Virtual Lymph Node”, in: “From the Laboratory to the Clinic, Book of Abstracts, Oct. 12-15, 1998, Seascape Resort, Aptos, Calif.”

Oligonucleotides may be used as adjuvants in conjunction with the Zika virus vaccine and/or immunogenic composition antigens and may contain two or more dinucleotide CpG motifs separated by at least three or more or even at least six or more nucleotides. CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) induce a predominantly Th1 response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.

Such oligonucleotide adjuvants may be deoxynucleotides. In certain embodiments, the nucleotide backbone in the oligonucleotide is phosphorodithioate, or a phosphorothioate bond, although phosphodiester and other nucleotide backbones such as PNA including oligonucleotides with mixed backbone linkages may also be used. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in U.S. Pat. Nos. 5,666,153, 5,278,302 and WO 95/26204.

Exemplary oligonucleotides have the following sequences. The sequences may contain phosphorothioate modified nucleotide backbones:

OLIGO 1:  (SEQ ID NO: 3) TCC ATG ACG TTC CTG ACG TT (CpG 1826); OLIGO 2:  (SEQ ID NO: 4) TCT CCC AGC GTG CGC CAT (CpG 1758);  OLIGO 3:  (SEQ ID NO: 5) ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG; OLIGO 4:  (SEQ ID NO: 6) TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006); and  OLIGO 5:  (SEQ ID NO: 7) TCC ATG ACG TTC CTG ATG CT (CpG 1668) 

Alternative CpG oligonucleotides include the above sequences with inconsequential deletions or additions thereto. The CpG oligonucleotides as adjuvants may be synthesized by any method known in the art (e.g., EP 468520). For example, such oligonucleotides may be synthesized utilizing an automated synthesizer. Such oligonucleotide adjuvants may be between 10-50 bases in length. Another adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 is disclosed in WO 00/09159.

Many single or multiphase emulsion systems have been described. One of skill in the art may readily adapt such emulsion systems for use with a Zika virus vaccine and/or immunogenic composition antigens so that the emulsion does not disrupt the antigen's structure. Oil in water emulsion adjuvants per se have been suggested to be useful as adjuvant compositions (EP 399 843B), also combinations of oil in water emulsions and other active agents have been described as adjuvants for vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Other oil emulsion adjuvants have been described, such as water in oil emulsions (U.S. Pat. No. 5,422,109; EP 0 480 982 B2) and water in oil in water emulsions (U.S. Pat. No. 5,424,067; EP 0 480 981 B).

The oil emulsion adjuvants for use with the Zika virus vaccines and/or immunogenic compositions described herein may be natural or synthetic, and may be mineral or organic. Examples of mineral and organic oils will be readily apparent to one skilled in the art.

In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system may include a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as “being capable of being transformed by metabolism” (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts (such as peanut oil), seeds, and grains are common sources of vegetable oils. Synthetic oils may also be used and can include commercially available oils such as NEOBEE® and others. Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and may be used with the Zika virus vaccine and/or immunogenic compositions. Squalene is a metabolizable oil virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619).

Exemplary oil emulsions are oil in water emulsions, and in particular squalene in water emulsions.

In addition, the oil emulsion adjuvants for use with the Zika virus vaccine and/or immunogenic compositions may include an antioxidant, such as the oil α-tocopherol (vitamin E, EP 0 382 271 B1).

WO 95/17210 and WO 99/11241 disclose emulsion adjuvants based on squalene, α-tocopherol, and TWEEN 80™, optionally formulated with the immunostimulants QS21 and/or 3D-MLA. WO 99/12565 discloses an improvement to these squalene emulsions with the addition of a sterol into the oil phase. Additionally, a triglyceride, such as tricaprylin (C27H5006), may be added to the oil phase in order to stabilize the emulsion (WO 98/56414).

The size of the oil droplets found within the stable oil in water emulsion may be less than 1 micron, may be in the range of substantially 30-600 nm, substantially around 30-500 nm in diameter, or substantially 150-500 nm in diameter, and in particular about 150 nm in diameter as measured by photon correlation spectroscopy. In this regard, 80% of the oil droplets by number may be within these ranges, more than 90% or more than 95% of the oil droplets by number are within the defined size ranges. The amounts of the components present in oil emulsions are conventionally in the range of from 2 to 10% oil, such as squalene; and when present, from 2 to 10% alpha tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. The ratio of oil:alpha tocopherol may be equal or less than 1 as this provides a more stable emulsion. SPAN 85™ may also be present at a level of about 1%. In some cases it may be advantageous that the Zika virus vaccines and/or immunogenic compositions disclosed herein will further contain a stabilizer.

The method of producing oil in water emulsions is well known to one skilled in the art. Commonly, the method includes the step of mixing the oil phase with a surfactant such as a PBS/TWEEN80® solution, followed by homogenization using a homogenizer, it would be clear to one skilled in the art that a method comprising passing the mixture twice through a syringe needle would be suitable for homogenizing small volumes of liquid. Equally, the emulsification process in microfluidizer (M110S microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted by one skilled in the art to produce smaller or larger volumes of emulsion. This adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.

Alternatively the Zika virus vaccines and/or immunogenic compositions may be combined with vaccine vehicles composed of chitosan (as described above) or other polycationic polymers, polylactide and polylactide-coglycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM.

Additional illustrative adjuvants for use in the Zika virus vaccines and/or immunogenic compositions as described herein include SAF (Chiron, Calif., United States), MF-59 (Chiron, see, e.g., Granoff et al. (1997) Infect Immun 65 (5):1710-1715), the SBAS series of adjuvants (e.g., SB-AS2 (an oil-in-water emulsion containing MLA and QS21); SBAS-4 (adjuvant system containing alum and MLA), available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (GlaxoSmithKline), RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560 (GlaxoSmithKline) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720.

Other examples of adjuvants include, but are not limited to, Hunter's TiterMax® adjuvants (CytRx Corp., Norcross, Ga.); Gerbu adjuvants (Gerbu Biotechnik GmbH, Gaiberg, Germany); nitrocellulose (Nilsson and Larsson (1992) Res. Immunol. 143:553-557); alum (e.g., aluminum hydroxide, aluminum phosphate) emulsion based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water emulsions, such as the Seppic ISA series of Montamide adjuvants (e.g., ISA-51, ISA-57, ISA-720, ISA-151, etc.; Seppic, Paris, France); and PROVAX® (IDEC Pharmaceuticals); OM-174 (a glucosamine disaccharide related to lipid A); Leishmania elongation factor; non-ionic block copolymers that form micelles such as CRL 1005; and Syntex Adjuvant Formulation. See, e.g., O'Hagan et al. (2001) Biomol Eng. 18(3):69-85; and “Vaccine Adjuvants: Preparation Methods and Research Protocols” D. O'Hagan, ed. (2000) Humana Press.

Other exemplary adjuvants include adjuvant molecules of the general formula: HO(CH₂CH₂O)n-A-R, (I) where, n is 1-50, A is a bond or —C(O)—, R is C1-50 alkyl or Phenyl C1-50 alkyl.

One embodiment consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), where n is between 1 and 50, 4-24, or 9; the R component is C1-50, C4-C20 alkyl, or C12 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, from 0.1-10%, or in the range 0.1-1%. Exemplary polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.

The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, an adjuvant combination may include the CpG as described above.

Further examples of suitable pharmaceutically acceptable excipients for use with the Zika virus vaccines and/or immunogenic compositions disclosed herein include water, phosphate buffered saline, isotonic buffer solutions.

Virus Purification

Further aspects of the present disclosure relate to methods of purifying Zika virus. In some embodiments, the method includes inoculating a plurality of cells with an inoculum containing a population of Zika viruses, and obtaining from one or more of the inoculated cells a Zika virus clonal isolate by plaque purification. In some embodiments, the cells are non-human cells (e.g., insect cells, mammalian cells, etc.). In some embodiments, the cells are insect cells (such as any of the mosquito cells/cell lines described herein). In some embodiments, the cells are mammalian cells (such as any of the mammalian cells/cell lines described herein). In some embodiments, the mammalian cells are monkey cells. In some embodiments, the mammalian cells are Vero cells.

In some embodiments, the population of Zika virus is heterogeneous (e.g., comprising two or more genotypes). The two or more genotypes differ from each other in at least one nucleotide. In some embodiments, the population of Zika viruses comprises a Zika virus clinical isolate (e.g., from strain PRVABC59) and/or one or more Zika viruses that have been previously passaged in cell culture. A clinical isolate of the Zika virus is obtained from a sample of a patient who is infected with Zika virus. In some embodiments, plaque purification (e.g., as described herein) allows for the substantial and/or complete separation of a (genetically homogenous) clonal isolate from a heterogeneous viral population. In some embodiments, the monkey cells are from a VERO cell line (e.g., VERO 10-87 cells). In some embodiments, the inoculum comprises human serum. In some embodiments, the inoculum comprises one or more adventitious agents (e.g., one or more contamination viruses). In some embodiments, plaque purification (e.g., as described herein) allows for the substantial and/or complete purification of a (genetically homogenous) clonal isolate away from one or more adventitious agents.

In some embodiments, the methods described for isolating and/or purifying a Zika virus clonal includes one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) additional plaque purifications of the Zika virus clonal isolate. In some embodiments, the methods described for isolating and/or purifying a Zika virus clonal isolate includes passaging the Zika virus clonal isolate one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) times in cell culture (e.g., in insect cells such as a mosquito cell line and/or in mammalian cells such as a VERO cell line).

Further aspects of the present disclosure relate to methods of purifying Zika virus for the preparation of a vaccine or immunogenic composition. In some embodiments, the methods include one or more (e.g., one or more, two or more, three or more, four or more, five or more, or six) steps of (in any order, including the following order): performing depth filtration of a sample or preparation containing a Zika virus; buffer exchanging and/or diluting a sample containing a Zika virus (e.g., by cross flow filtration (CFF)) to produce a retentate; binding a sample comprising a Zika virus to an ion exchange membrane (e.g., an anion exchange membrane, a cation exchange membrane) to produce a bound fraction, where the bound fraction comprises the Zika virus, and eluting the bound fraction from the ion exchange membrane; treating a sample containing a Zika virus with an effective amount of any of the chemical inactivators described herein; neutralizing a sample containing a chemically inactivated Zika virus with sodium metabisulfite; and/or purifying a neutralized sample comprising a chemically inactivated Zika virus (e.g., by cross flow filtration (CFF)). In some embodiments, the method includes the steps of (a) passing a sample containing a Zika virus through a first depth filter to produce a first eluate, where the first eluate contains the Zika virus; (b) buffer exchanging and/or diluting the first eluate by cross flow filtration (CFF) to produce a first retentate, where the first retentate contains the Zika virus; (c) binding the first retentate to an ion exchange membrane to produce a first bound fraction, where the first bound fraction contains the Zika virus, and eluting the first bound fraction from the ion exchange membrane to produce a second eluate, where the second eluate contains the Zika virus; (d) passing the second eluate through a second depth filter to produce a second retentate, wherein the second retentate contains the Zika virus; (e) treating the second retentate with an effective amount of a chemical inactivator; (f) neutralizing the treated second retentate with sodium metabisulfite; and (g) purifying the neutralized second retentate by cross flow filtration (CFF).

Depth filters may be applied in a cartridge or capsule format, such as with the SUPRACAP™ series of depth filter capsules (Pall Corporation) using a Bio 20 SEITZ® depth filter sheet. Other suitable depth filtration techniques and apparatuses are known in the art and include Sartorius PP3 filters. In some embodiments, the depth filter has a pore size of between about 0.2 μm and about 3 μm. In some embodiments, the pore size of the depth filter is less than about any of the following pore sizes (in μm): 3, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, and 0.4. In some embodiments, the pore size of the depth filter is greater than about any of the following pore sizes (in μm): 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, or 2.8. That is, the pore size of the depth filter can be any of a range of pore sizes (in μm) having an upper limit of 3, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, and 0.4 and an independently selected lower limit of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, or 2.8; wherein the lower limit is less than the upper limit.

As described herein, cation exchange and anion exchange chromatography may be used in the methods of the present disclosure to purify a Zika virus harvested from a cell of the present disclosure. For example, clarified viral harvest may be basified, loaded onto an anion exchange membrane, eluted by salt or pH, filtered, and inactivated. This is only an exemplary scheme, and one of skill in the art may readily contemplate variants thereof with substituted, deleted, inserted, or reordered steps.

Anion and cation exchange chromatography both rely on the attraction of charged macromolecules of interest (e.g., a virus) in a mobile phase to a substrate having an opposite charge. In cation exchange chromatography, the negatively charged substrate or membrane attracts positively charged macromolecules. In anion exchange chromatography, the positively charged substrate or membrane attracts negatively charged macromolecules. Once macromolecules are bound or loaded onto the substrate, they may be eluted in linear or step-wise fashion from the substrate in a manner dependent on their characteristics, thereby enacting a separation of differently charged molecules. This principle may be used to purify viruses from other macromolecules. Elution may be effected by varying pH or salt content of the mobile phase buffer. Elution may be gradient or step-wise. As described herein, elution may be effected using a change in pH of the mobile phase or by using a change in ionic strength of the mobile phase (e.g., through addition of a salt). A variety of salts are used for elution, including without limitation sodium chloride, potassium chloride, sodium sulphate, potassium sulphate, ammonium sulphate, sodium acetate, potassium phosphate, calcium chloride, and magnesium chloride. In certain embodiments, the salt is NaCl. A variety of suitable buffers are known in the art and described herein. Viral purification methods using ion exchange chromatography are also generally known; see, e.g., purification of influenza virus available online at www.pall.com/pdfs/Biopharmaceuticals/MustangQXT_AcroPrep_USD2916.pdf.

A variety of devices known in the art are suitable for cation exchange chromatography (optionally including filtration), such as the Mustang® S system (Pall Corporation), which uses a cation exchange membrane with a 0.65 μm pore size. A variety of functional groups are used for cation exchange membranes, including without limitation pendant sulfonic functional groups in a cross-linked, polymeric coating. A variety of buffers may be used to bind an eluate containing a Zika virus of the present disclosure to a cation exchange membrane. Exemplary buffers include, without limitation, citrate and phosphate buffers (additional buffers are described infra). In some embodiments, a buffer used in cation exchange chromatography (e.g., in loading and/or elution) contains polysorbate (e.g., TWEEN®-80 at 0.05%, 0.1%, 0.25%, or 0.5%).

A variety of devices known in the art are suitable for anion exchange chromatography (optionally including filtration), such as the Mustang® Q system (Pall Corporation), which uses an anion exchange membrane with a 0.8 μm pore size. Another suitable anion exchange membrane is SartobindQ IEXNano. A variety of functional groups are used for anion exchange membranes, including without limitation pendant quaternary amine functional groups in a cross-linked, polymeric coating. A variety of buffers may be used to bind an eluate containing a Zika virus of the present disclosure to an anion exchange membrane. Exemplary buffers include, without limitation, phosphate buffer (additional buffers are described infra). In some embodiments, a buffer used in anion exchange chromatography (e.g., in loading and/or elution) contains polysorbate (e.g., TWEEN®-80 at 0.05%, 0.1%, 0.25%, or 0.5%). In some embodiments, the virus is eluted by step elution, e.g. using 250 mM NaCl, 500 mM NaCl and 750 mM NaCl.

Formulations and Dose of Vaccines and/or Immunogenic Compositions

Further aspects of the present disclosure relate to formulations of vaccines and/or immunogenic compositions of the present disclosure containing one or more antigens from a Zika virus described herein.

Such vaccines and/or immunogenic compositions of the present disclosure containing one or more antigens from a Zika virus described herein may be useful for treating or preventing Zika virus infection in a human subject in need thereof and/or inducing an immune response, such as a protective immune response, against Zika virus in a human subject in need thereof.

Typically, vaccines and/or immunogenic compositions of the present disclosure are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. Such preparations may also be emulsified or produced as a dry powder. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, sucrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine or immunogenic composition may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine or immunogenic composition.

Vaccines or immunogenic compositions may be conventionally administered parenterally, by injection, for example, either subcutaneously, transcutaneously, intradermally, subdermally or intramuscularly. In certain embodiments the composition is administered intramuscular or subcutaneously. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral, peroral, intranasal, buccal, sublingual, intraperitoneal, intravaginal, anal and intracranial formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, or even 1-2%. In certain embodiments, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the Zika virus vaccine and/or immunogenic composition described herein is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into conveniently sized molds and allowed to cool and to solidify.

Formulations suitable for intranasal delivery include liquids (e.g., aqueous solution for administration as an aerosol or nasal drops) and dry powders (e.g. for rapid deposition within the nasal passage). Formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, sucrose, trehalose, xylitol, and chitosan. Mucosadhesive agents such as chitosan can be used in either liquid or powder formulations to delay mucociliary clearance of intranasally-administered formulations. Sugars such as mannitol, sorbitol, trehalose, and/or sucrose can be used as stability agents in liquid formulations and as stability, bulking, or powder flow and size agents in dry powder formulations. In addition, adjuvants such as monophosphoryl lipid A (MLA), or derivatives thereof, or CpG oligonucleotides can be used in both liquid and dry powder formulations as an immunostimulatory adjuvant.

Formulations suitable for oral delivery include liquids, solids, semi-solids, gels, tablets, capsules, lozenges, and the like. Formulations suitable for oral delivery include tablets, lozenges, capsules, gels, liquids, food products, beverages, nutraceuticals, and the like. Formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, sorbitol, trehalose, polyols such as sugars such as sucrose, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Other Zika virus vaccines and immunogenic compositions may take the form of solutions, suspensions, pills, sustained release formulations or powders and contain 10-95% of active ingredient, or 25-70%. For oral formulations, cholera toxin is an interesting formulation partner (and also a possible conjugation partner).

The Zika virus vaccines and/or immunogenic compositions when formulated for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foams or sprays. Any of the foregoing formulations may contain agents in addition to Zika virus vaccine and/or immunogenic compositions, such as carriers, known in the art to be appropriate.

In some embodiments, the Zika virus vaccines and/or immunogenic compositions of the present disclosure may be formulated for systemic or localized delivery. Such formulations are well known in the art. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Systemic and localized routes of administration include, e.g., intradermal, topical application, intravenous, intramuscular, etc.

The vaccines and/or immunogenic compositions of the present disclosure may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The dosage of the antigen may range in particular from, about 1 μg to about 100 μg, about 1 μg to about 40 μg, about 1 μg to about 30 μg, about 1 μg to about 20 μg, about 1 μg to about 15 μg, or from about 2 μg to about 15 μg, or from about 5 μg to about 15 μg, or from about 10 μg to about 15 μg, The dosage may in particular be about 2 μg, about 5 μg, about 10 μg, about 15 μg or about 20 μg, in particular about 10 μg. The amount of the antigen, i.e. the purified inactivated Zika virus, can be determined by a Bradford assay (Bradford et al. (1976) Anal. Biochem. 72: 248-254) using defined amounts of recombinant Zika envelope protein to establish the standard curve. Thus, the dosage of the antigen may also be referred to as micrograms (μg) of Zika Envelope protein E (μg Env). μg Antigen and μg Env thus mean the same within the meaning of this disclosure. In some embodiments, the vaccines and/or immunogenic compositions contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form of a purified inactivated whole Zika virus such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1 as described herein.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2.

In some embodiments, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form of a purified inactivated whole plaque purified Zika virus isolate comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2.

In certain such embodiments the vaccine or immunogenic composition comprises

a dose of about 10 μg of purified inactivated whole virus

about 200 μg aluminum hydroxide,

a buffer; and optionally

a sugar such as sucrose.

Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine or immunogenic composition are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like for e.g. intramuscular or subcutaneous administration. The dosage of the vaccine or immunogenic composition will depend on the route of administration and may vary according to the age of the person to be vaccinated and the formulation of the antigen. The vaccine or immunogenic composition can have a unit dosage volume of more than 0.5 mL, of 0.5 mL or of less than 0.5 mL, as described herein. For instance, it can be administered at a volume of 0.25 mL. A volume of 0.5 mL are suitable for intramuscular or subcutaneous administration.

Delivery agents that improve mucoadhesion can also be used to improve delivery and immunogenicity especially for intranasal, oral or lung based delivery formulations. One such compound, chitosan, the N-deacetylated form of chitin, is used in many pharmaceutical formulations. It is an attractive mucoadhesive agent for intranasal vaccine delivery due to its ability to delay mucociliary clearance and allow more time for mucosal antigen uptake and processing. In addition, it can transiently open tight junctions which may enhance transepithelial transport of antigen to the NALT. In a recent human trial, a trivalent inactivated influenza vaccine administered intranasally with chitosan but without any additional adjuvant yielded seroconversion and HI titers that were only marginally lower than those obtained following intramuscular inoculation.

Chitosan can also be formulated with adjuvants that function well intranasally such as the genetically detoxified E. coli heat-labile enterotoxin mutant LTK63. This adds an immunostimulatory effect on top of the delivery and adhesion benefits imparted by chitosan resulting in enhanced mucosal and systemic responses.

Finally, it should be noted that chitosan formulations can also be prepared in a dry powder format that has been shown to improve vaccine stability and result in a further delay in mucociliary clearance over liquid formulations. This was seen in a recent human clinical trial involving an intranasal dry powder diphtheria toxoid vaccine formulated with chitosan in which the intranasal route was as effective as the traditional intramuscular route with the added benefit of secretory IgA responses. The vaccine was also very well tolerated. Intranasal dry powdered vaccines for anthrax containing chitosan and MLA, or derivatives thereof, induce stronger responses in rabbits than intramuscular inoculation and are also protective against aerosol spore challenge.

Intranasal vaccines represent an exemplary formulation as they can affect the upper and lower respiratory tracts in contrast to parenterally administered vaccines which are better at affecting the lower respiratory tract. This can be beneficial for inducing tolerance to allergen-based vaccines and inducing immunity for pathogen-based vaccines.

In addition to providing protection in both the upper and lower respiratory tracts, intranasal vaccines avoid the complications of needle inoculations and provide a means of inducing both mucosal and systemic humoral and cellular responses via interaction of particulate and/or soluble antigens with nasopharyngeal-associated lymphoid tissues (NALT).

Vaccines and/or immunogenic compositions of the present disclosure are pharmaceutically acceptable. They may include components in addition to the antigen and adjuvant, e.g. they will typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccines and/or immunogenic compositions of the present disclosure may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range.

The pH of a vaccine or immunogenic composition will generally be between 5.0 and 8.5 or 5.0 and 8.1, and more typically between 6.0 and 8.5 e.g. between 6.0 and 8.0, between 6.5 and 8.0, between 6.5 and 7.5, between 7.0 and 8.5, between 7.0 and 8.0, or between 7.0 and 7.8. A manufacturing process of the present disclosure may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.

The vaccine or immunogenic composition is preferably sterile. It is preferably non pyrogenic, e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. It is preferably gluten free.

In certain embodiments, the vaccines and/or immunogenic compositions of the present disclosure may include a detergent in an effective concentration. In some embodiments, an effective amount of detergent may include without limitation, about 0.00005% v/v to about 5% v/v or about 0.0001% v/v to about 1% v/v. In certain embodiments, an effective amount of detergent is about 0.001% v/v, about 0.002% v/v, about 0.003% v/v, about 0.004% v/v, about 0.005% v/v, about 0.006% v/v, about 0.007% v/v, about 0.008% v/v, about 0.009% v/v, or about 0.01% v/v. Without wishing to be bound by theory, detergents help maintain the vaccines and/or immunogenic compositions of the present disclosure in solution and help to prevent the vaccines and/or immunogenic compositions from aggregating.

Suitable detergents include, for example, polyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), octoxynol (such as octoxynol-9 (Triton X 100) or t-octylphenoxypolyethoxyethanol), cetyl trimethyl ammonium bromide (‘CTAB’), and sodium deoxycholate, particularly for a split or surface antigen vaccine. The detergent may be present only at trace amounts. Other residual components in trace amounts could be antibiotics (e.g. neomycin, kanamycin, polymyxin B). In some embodiments, the detergent contains polysorbate. In some embodiments, the effective concentration of detergent includes ranges from about 0.00005% v/v to about 5% v/v.

The vaccines and/or immunogenic compositions are preferably stored at between 2° C. and 8° C. They should ideally be kept out of direct light. The antigen and emulsion will typically be in admixture, although they may initially be presented in the form of a kit of separate components for extemporaneous admixing. Vaccines and/or immunogenic compositions will generally be in aqueous form when administered to a human subject.

Methods of the Present Disclosure

The present invention is in particular also directed to a method of treating or preventing, in particular preventing Zika virus infection and/or preventing Zika virus disease in a human subject in need thereof comprising administering to a human subject or a human subject population a therapeutically effective amount of a vaccine or immunogenic composition as described herein.

The disease is in general mild and of short duration. Some clinical manifestations include, but are not limited to, mild fever, maculopapular rash, conjunctivitis and arthralgia. Despite mild clinical symptoms in the pregnant woman, Zika virus infection during pregnancy has been associated with serious outcomes for the fetus and newborn. The severity of the disease is related to the consequences in the fetus and newborn child from women with Zika virus infection during pregnancy. The spectrum of congenital anomalies associated with Zika virus infection, known as Congenital Zika Syndrome (CZS), consists of severe microcephaly with partially collapsed skull, cerebral cortices with subcortical calcifications, macular scarring and focal pigmentary retinal mottling, congenital contractures, and marked early hypertonia with symptoms of extrapyramidal involvement. Furthermore the Zika virus is a neurotropic flavivirus that can potentially cause disease within the central nervous system. There is additionally a Worldwide concern over Zika virus causing Guillain-Barré Syndrome (GBS).

The prevention of the Zika virus disease thus does not only concern the human subject being treated but extends to the fetus and newborn in case the human subject being treated is or will be pregnant. The method according to the invention thus comprises treating the human subject by administering to the human subject the vaccine or immunogenic composition and the treating of the fetus and newborn by administering to a pregnant human subject or a human subject that intends to become pregnant or woman of childbearing potential the vaccine or immunogenic composition.

Further aspects of the present disclosure relate to methods for using vaccines and/or or immunogenic compositions described herein containing one or more antigens from at least one Zika virus (e.g., a clonal Zika virus isolate, a Zika virus comprising a non-human cell adaptation mutation such as a non-human cell adaptation mutation in protein NS1) to treat or prevent Zika virus in a human subject in need thereof and/or to induce an immune response to Zika virus in a human subject in need thereof.

In certain such methods, the vaccines and/or immunogenic compositions contains a dose of 1 μg to 15 μg, or 2 μg, or 5 μg, or 10 μg of a purified inactivated whole Zika virus such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1 as described herein optionally in combination with one or more adjuvants, such as 100 μg to about 300 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole plaque purified Zika virus isolate comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the present disclosure relates to methods for treating or preventing Zika virus infection in a human subject in need thereof by administering to the human subject a therapeutically effective amount of a vaccine and/or immunogenic composition of the present disclosure containing one or more antigens from at least one Zika virus (e.g., a clonal Zika virus isolate, a Zika virus comprising a non-human cell adaptation mutation such as a non-human cell adaptation mutation in protein NS1). In some embodiments, the present disclosure relates to methods for inducing an immune response to Zika virus in a human subject in need thereof by administering to the human subject a therapeutically effective amount of a vaccine and/or immunogenic composition of the present disclosure containing one or more antigens from at least one Zika virus (e.g., a clonal Zika virus isolate, a Zika virus comprising a non-human cell adaptation mutation such as a non-human cell adaptation mutation in protein NS1). In some embodiments, the administering step induces a protective immune response against Zika virus in the human subject. In some embodiments, the human subject is pregnant or intends to become pregnant or woman of childbearing potential.

The Zika virus vaccines and/or immunogenic compositions disclosed herein may be used to protect or treat a human subject susceptible to, or suffering from a viral infection, by means of administering the vaccine by intranasal, peroral, oral, buccal, sublingual, intramuscular, intraperitoneal, intradermal, transdermal, subdermal, intravaginal, anal, intracranial, intravenous, transcutaneous, or subcutaneous administration, in particular intramuscular administration. Methods of systemic administration of the vaccines and/or immunogenic compositions of the present disclosure may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961), or needleless pressure liquid jet device (U.S. Pat. Nos. 4,596,556; 5,993,412), or transdermal patches (WO 97/48440; WO 98/28037). The Zika virus vaccines and/or immunogenic compositions of the present disclosure may also be applied to the skin (transdermal or transcutaneous delivery WO 98/20734; WO 98/28037). The Zika virus vaccines and/or immunogenic compositions of the present disclosure therefore may include a delivery device for systemic administration, pre-filled with the Zika virus vaccine or immunogenic compositions. Accordingly there is provided methods for treating or preventing Zika virus infection and/or for inducing an immune response in a human subject, including the step of administering a vaccine or immunogenic composition of the present disclosure and optionally including an adjuvant and/or a carrier, to the human subject, where the vaccine or immunogenic composition is administered via the parenteral or systemic route.

The vaccines and/or immunogenic compositions of the present disclosure may be used to protect or treat a human subject susceptible to, or suffering from a viral infection, by means of administering the vaccine or immunogenic composition via a mucosal route, such as the oral/alimentary or nasal route. Alternative mucosal routes are intravaginal and intra-rectal. The mucosal route of administration may be via the nasal route, termed intranasal vaccination. Methods of intranasal vaccination are well known in the art, including the administration of a droplet, spray, or dry powdered form of the vaccine into the nasopharynx of the individual to be immunized Nebulized or aerosolized vaccine formulations are potential forms of the Zika virus vaccines and/or immunogenic compositions disclosed herein. Enteric formulations such as gastro resistant capsules and granules for oral administration, suppositories for rectal or vaginal administration are also formulations of the vaccines and/or immunogenic compositions of the present disclosure.

The Zika virus vaccines and/or immunogenic compositions of the present disclosure may also be administered via the oral route. In such cases the pharmaceutically acceptable excipient may also include alkaline buffers, or enteric capsules or microgranules. The Zika virus vaccines and/or immunogenic compositions of the present disclosure may also be administered by the vaginal route. In such cases, the pharmaceutically acceptable excipients may also include emulsifiers, polymers such as CARBOPOL®, and other known stabilizers of vaginal creams and suppositories. The Zika virus vaccines and/or immunogenic compositions may also be administered by the rectal route. In such cases the excipients may also include waxes and polymers known in the art for forming rectal suppositories.

In some embodiments, the administering step includes one or more administrations. Administration can be by a single dose schedule or a multiple dose (prime-boost) schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Typically they will be given by the same route, such as by intramuscular or subcutaneous administration. Multiple doses will typically be administered at least 1 week apart (e.g. about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 16 weeks, etc., such as 1 to 16 weeks apart). Giving two doses separated by from 25-30 days (e.g. 28 days, 4 weeks) is particularly useful. In certain such embodiments the mode of administration is intramuscular or subcutaneous administration.

The methods of the present disclosure include administration of a therapeutically effective amount or an immunogenic amount of the Zika virus vaccines and/or immunogenic compositions of the present disclosure. A therapeutically effective amount or an immunogenic amount may be an amount of the vaccines and/or immunogenic compositions of the present disclosure that will induce a protective immunological response in the uninfected, infected or unexposed human subject to which it is administered. Such a response will generally result in the development in the human subject of a secretory, cellular and/or antibody-mediated immune response to the vaccine. Usually, such a response includes, but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell.

In certain such methods, the vaccines and/or immunogenic compositions contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus such as a Zika virus with a mutation which is a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1 as described herein optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivated whole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition contains a dose of 1 μg to 15 μg, or 2 μg, or 5 μg or 10 μg of a purified inactivated whole plaque purified Zika virus isolate comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:1, wherein the Zika virus is derived from strain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2 optionally in combination with one or more adjuvants, such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods the administration of the vaccine or immunogenic composition induces the generation of neutralizing antibody titers to Zika virus in a human subject of greater than 10, or greater than 50, or greater than 100, or greater than 200 or greater than 1000, or greater than 1500, or greater than 2000, or greater than 2000, or greater than 3000, as determined by the plaque reduction neutralization test (PRNT)

In certain such methods the administration of the vaccine or immunogenic composition induces the generation of neutralizing antibody titers to Zika virus in a human subject of greater than 300 or greater than 500, or greater than 1000, or greater than 1500, or greater than 2000, or greater than 2000, or greater than 3000, or greater than 5000, or greater than 10,000, as determined by the reporter virus particle neturalization assay (RVP).

In certain such methods the above neutralizing antibody titers are achieved 14 and/or 28 days after the administration.

In certain such methods 14 and/or 28 days after the administration of the vaccine or immunogenic composition the generation of neutralizing antibody titers to Zika virus in a human subject is greater than 250, as determined by the plaque reduction neutralization test (PRNT)

In certain such methods 14 and/or 28 days after the administration of the vaccine or immunogenic composition the generation of neutralizing antibody titers to Zika virus in a human subject is greater than 1000, as determined by the reporter virus particle neturalization assay (RVP).

In certain such methods such titers are achieved 14 and/or 28 days after the administration. Such generation of neutralizing antibodies provides for high seroconversion rates in a Zika virus seronegative population of at least 20 human subjects. In certain such embodiments the seroconversion rate is at least 60%, at least 70%, at least 80%, at least 90% at least 95% or 100%.

In certain such methods such titers are achieved 14 and/or 28 days after the administration. Such generation of naturalizing antibodies provides for high seropositvity rates in a population, in particular in a Zika virus seronegative population, of at least 20 human subjects. In certain such embodiments the seropositvity rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

According to the inventive method the vaccine or immunogenic composition is administered as a single dose or as multiple doses as e.g. in a first (prime) and a second (boost) administration as e.g. administered 1 to 16 weeks apart. In certain such methods the second (boost) administration is administered at least 28 days after the first (prime) administration.

In certain such methods 14 and/or 28 days after the administration of a single dose or of multiple doses as e.g. in a first (prime) and after a second (boost) administration, a high seroconversion rate is achieved in a Zika virus seronegative population, of at least 20 human subjects. In certain such embodiments the seroconversion rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In certain such methods including the administration as first (prime) and second (boost) administration 14 and/or 28 days after the second (boost) administration a high seroconversion rate is achieved in a Zika virus seronegative population, of at least 20 human subjects. In certain such embodiments the seroconversion rate is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In certain such methods 14 and/or 28 days after the administration as single dose or after the first (prime) administration a high seroconversion rate is achieved in a Zika virus seronegative population, of at least 20 human subjects. In certain such embodiments the seroconversion rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In certain such methods 14 and/or 28 days after the administration of a single dose or of multiple doses as e.g. in a first (prime) and after a second (boost) administration, a high seropositivity rate is achieved in a population, in particular in a Zika virus seronegative population or in a Flavivirus naïve population, of at least 20 human subjects. In certain such embodiments the seropositvity rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In certain such methods including the administration as first (prime) and second (boost) administration 14 and/or 28 days after the second (boost) administration a high seropositivity rate is achieved in a population, in particular in a Zika virus seronegative population, of at least 20 human subjects. In certain such embodiments the seropositivity rate is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In certain such methods 14 and/or 28 days after the administration as single dose or after the first (prime) administration a high seropositivity rate is achieved in a population, in particular in a Zika virus seronegative population or in a Flavivirus naïve population, of at least 20 human subjects. In certain such embodiments the seropositivity rate is 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In certain such methods 14 and/or 28 days after the single dose or prime administration of the vaccine or immunogenic composition the neutralizing antibody titers to Zika virus in a human subject, in particular a flavivirus naive human subject, or in particular in a Zika virus seronegative human subject, are greater than 200, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the single dose or prime administration of the vaccine or immunogenic composition the geometric mean neutralizing antibody titers to Zika virus in particular in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects are greater than 200, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the single dose or prime administration of the vaccine or immunogenic composition the neutralizing antibody titers to Zika virus in a human subject, in particular a flavivirus naive human subject, or in particular in a Zika virus seronegative human subject, are greater than 1000, as determined by the reporter virus particle neutralization assay (RVP).

In certain such methods 14 and/or 28 days after the single dose or prime administration of the vaccine or immunogenic composition the geometric mean neutralizing antibody titers to Zika virus in particular in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects are greater than 1000, as determined by the reporter virus particle neutralization assay (RVP).

Such generation of neutralizing antibodies provides for a high seroconversion rates in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects after the single dose or prime administration. In certain such embodiments the seroconversion rate is 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

Such generation of neutralizing antibodies provides for high seropositvity rates in a population, in particular in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects after the single dose or prime administration. In certain such embodiments the seropositvity rate is 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

In certain methods the administration comprises prime and boost administration wherein the prime and boost administration takes place from about 1 to about 16 weeks apart. In certain such methods the second (boost) administration is administered at least 28 days after the first (prime) administration.

In certain such methods 14 and/or 28 days after the boost administration of the vaccine or immunogenic composition the neutralizing antibody titers to Zika virus in a human subject, in particular a flavivirus naive human subject or in particular in a Zika virus seronegative human subject, are greater than 1000, or greater than 1500, or greater than 3000, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the boost administration of the vaccine or immunogenic composition the geometric mean neutralizing antibody titers to Zika virus in particular in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects are greater than 1000, or greater than 1500, or greater than 3000, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the boost administration of the vaccine or immunogenic composition the neutralizing antibody titers to Zika virus in a human subject, in particular a flavivirus naive human subject or in particular in a Zika virus seronegative human subject, are greater than 3000, or greater than 5000, or greater than 10000, as determined by the reporter virus particle neutralization assay (RVP).

In certain such methods 14 and/or 28 days after the boost administration of the vaccine or immunogenic composition the geometric mean neutralizing antibody titers to Zika virus in particular in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects are greater than 3000, or greater than 5000, or greater than 10000, as determined by the reporter virus particle neutralization assay (RVP).

Such generation of neutralizing antibodies provides for a high seroconversion rates in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects after the boost administration. In certain such embodiments the seroconversion rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%

Such generation of neutralizing antibodies provides for high seropositvity rates in a population, in particular in a flavivirus naive population, of at least 20 human subjects after the boost administration. In certain such embodiments the seropositvity rate is 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%

Such generation of neutralizing antibodies provides for a high seroconversion rates in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects after the single dose or prime administration. In certain such embodiments the seroconversion rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

Such generation of neutralizing antibodies provides for a high seroconversion rates in a flavivirus naive population or in particular in a Zika virus seronegative population of at least 20 human subjects after the single dose or prime administration. In certain such embodiments the seropositivity rate is at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

Furthermore, certain aspects of the present disclosure relate to a method of treating or preventing, in particular preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a single dose or as multiple doses as e.g. in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the administration induces systemic side effects in less than 50% of a human subject population of at least 20 human subjects, in particular in a population of at least 20 flavivirus naïve human subjects or in particular in a population of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the administration induces headache symptoms in less than 29% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the administration induces fever in 4% or less, and/or fatigue in 33% or less, and/or arthralgia in 10% or less, and/or myalgia in 17% or less, and/or malaise in 15% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces systemic side effects in less than 40% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces

-   -   at least 40% less, or at least 45% less fatigue, and/or     -   no more fever, and/or     -   no more, or at least 10% less, or at least 20% less, or at least         25% less myalgia, and/or     -   no more, or at least 10% less, or at least 20% less malaise         compared to 7 days after the prime administration in a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.

The “%-decrease of adverse events (AEs) after boost administration compared to after prime administration” is defined by the following equation:

${\% \mspace{14mu} {decrease}} = {\frac{\begin{pmatrix} {{\% \mspace{14mu} {subjects}\mspace{14mu} {experiencing}\mspace{14mu} {AEs}\mspace{14mu} {after}\mspace{14mu} {prime}} -} \\ {\% \mspace{14mu} {subjects}\mspace{14mu} {experiencing}\mspace{14mu} {AEs}\mspace{14mu} {after}\mspace{14mu} {boost}} \end{pmatrix}}{\% \mspace{14mu} {subjects}\mspace{14mu} {experiencing}\mspace{14mu} {AEs}\mspace{14mu} {after}\mspace{14mu} {prime}} \times 100}$

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fever in less than 4% or in 0% of a human subject population of at least 20 flavivirus naïve human subjects or at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fatigue in less than 19% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces myalgia in less than 12% or less than 8% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces malaise in less than 13% or in 10% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces headache symptoms in 20% or less, and arthralgia in 8% or less, and fever in less than 4%, and fatigue in less than 19%, and myalgia in less than 12%, and malaise in less than 13% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition comprises a dose of 1 μg to 40 μg of the (one) antigen, wherein the antigen is an inactivated whole virus.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as a single dose administration or multi dose administration including at least a first (prime) and a second (boost) administration wherein the vaccine or immunogenic composition comprises a dose of about 5 μg of purified inactivated whole virus.

According to certain aspects of the present disclosure the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces systemic side effects in less than 50%, or in less than 45%, or in less than 40% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces systemic side effects in less than 40%, or in less than 35%, or in less than 30%, or in less than 25%, or in less than 20%, or in less than 15% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fever in less than 3%, or 0%, of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fever in less than 4%, or in less than 3%, or 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces headache in less than 29% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces headache in less than 20%, or in less than 15%, or in less than 10%, or in less than 5% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fatigue in less than 30%, or in less than 25%, or in less than 20% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fatigue in less than 20%, or in less than 15%, or in less than 10% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces arthralgia in less than 4% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces arthralgia in less than 5%, or in less than 2%, or in 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces myalgia in 17% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces myalgia in less than 12%, or in less than 10%, or in less than 5% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces malaise in less than 10% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces malaise in less than 14%, or in less than 10%, or in less than 5%, or 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces

-   -   at least 70% less, or at least 60% less, or at least 50% less,         or at least 40% less, or at least 35% less, or at least 30% less         systemic side effects, and/or     -   no increase in fever, and/or     -   at least 80% less, or at least 70% less, or at least 60% less,         or at least 50% less, or at least 45% less headache, and/or     -   at least 60% less, or at least 55% less, or at least 50% less,         or at least 45% less, or at least 40% less fatigue, and/or     -   no increase in arthralgia, or at least 80% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less arthralgia, and/or     -   no increase in myalgia, or at least 70% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less myalgia, and/or     -   no increase in malaise, or at least 80% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less malaise compared to 7 days after the prime         administration in a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.

According to certain aspects of the present disclosure the vaccine or immunogenic composition is administered as a single dose administration or multi dose administration including at least a first (prime) and a second (boost) administration wherein the vaccine or immunogenic composition comprises a dose of about 10 μg of purified inactivated whole virus.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces systemic side effects in less than 50% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces systemic side effects in less than 40%, or in less than 35%, or in less than 30% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fever in less than 4% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fever in less than 4%, or in less than 3%, or 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces headache in less than 29%, or in less than 25%, or in less than 20%, or in less than 15% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces headache in 20% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fatigue in 33% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fatigue in less than 20% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces arthralgia in 10% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces arthralgia in less than 5% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces myalgia in 17% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces myalgia in less than 12%, or in less than 10% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces malaise in 15% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces malaise in less than 13%, or in 10% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces

-   -   at least 40% less, or at least 35% less, or at least 30% less,         or at least 25% less systemic side effects, and/or     -   no increase in fever, or at least 80% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less fever, and/or     -   at least 45% less, or at least 40% less fatigue, and/or     -   no increase in arthralgia, or at least 65% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less arthralgia, and/or     -   no increase in myalgia, or at least 45% less, or at least 40%         less, or at least 20% less, or at least 10% less myalgia, and/or     -   no increase in malaise, or at least 20% less, or at least 10%         less malaise         compared to 7 days after the prime administration in a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.

According to certain such aspects of the present disclosure the vaccine or immunogenic composition is administered as a single dose administration or multi dose administration including at least a first (prime) and a second (boost) administration wherein the vaccine or immunogenic composition comprises a dose of about 2 μg of purified inactivated whole virus.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces systemic side effects in less than 50%, or in less than 45%, or in less than 40%, or in less than 35% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces systemic side effects in less than 40%, or in less than 35% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fever in less than 3%, or 0%, of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fever in less than 4%, or in less than 3%, or 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces headache in less than 29%, or in less than 25%, or in less than 20% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces headache in less than 20%, or in less than 15% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces fatigue in less than 30%, or in less than 25% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fatigue in less than 20%, or in less than 15% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces arthralgia in less than 4% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces arthralgia in less than 8% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces myalgia in 17% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces myalgia in less than 12%, or in less than 10% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the administration of the vaccine or immunogenic composition until 7 days after the single dose or prime administration induces malaise in less than 10% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects and/or the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces malaise in less than 13%, or in less than 10%, or in less than 5%, or 0% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces

-   -   no increase in fever, and/or     -   at least 50% less, or at least 45% less, or at least 40% less         fatigue, and/or     -   no increase in myalgia, or at least 20% less, or at least 10%         less myalgia, and/or     -   no increase in malaise, or at least 80% less, or at least 60%         less, or at least 40% less, or at least 20% less, or at least         10% less malaise         compared to 7 days after the prime administration in a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.

According to a certain method the human subject or human subject population is from a Zika endemic region. According to a certain such method the human subject or human subject population is from a Zika endemic region subject to an outbreak. According to a certain such method the human subject or human subject population is flavivirus naïve.

According to a certain method the human subject or human subject population is from a Zika non-endemic region. According to a certain such method the human subject or human subject population is from a Zika non-endemic region travelling to an endemic region. According to a certain such method the human subject or human subject population is flavivirus naïve.

According to a certain method the human subject or human subject population is Hispanic, Latino, American Indian, Alaska native, Asian, Black or African American, Native Hawaiian or White or a mixture thereof.

According to a certain method the human subject or human subject population is 18 to 29 years of age.

According to a certain method the human subject or human subject population is 30 to 49 years of age.

The vaccine or immunogenic composition as disclosed herein is also disclosed for use in a method as disclosed herein.

Also disclosed herein is the use of the vaccine or immunogenic composition as disclosed herein in the manufacture of a medicament for the method as disclosed herein.

In some embodiments, the protective immunological response induced in the human subject after administration of a vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure is greater than the immunological response induced in a corresponding human subject administered a vaccine and/or immunogenic composition containing a Zika virus that is not adapted for non-human cell growth and/or comprises a different non-human cell adaptation mutation. In some embodiments, the protective immunological response induced in the human subject after administration of the vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% greater than the immunological response induced in a corresponding human subject administered a vaccine and/or immunogenic composition containing a Zika virus that is not adapted for non-human cell growth and/or comprises a different non-human cell adaptation mutation. Methods of measuring protective immunological responses are generally known to one of ordinary skill in the art.

In some embodiments, administration of a vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure induces generation of neutralizing antibodies to Zika virus in the human subject. In some embodiments, administration of a vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure induces generation of neutralizing antibodies to Zika virus in the human subject in an amount that is greater than the amount of neutralizing antibodies induced in a corresponding human subject administered a vaccine and/or immunogenic composition containing a Zika virus that is not adapted for non-human cell growth and/or comprises a different non-human cell adaptation mutation. In some embodiments, administration of a vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure induces generation of neutralizing antibodies to Zika virus in the human subject in an amount that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% greater than the than the amount of neutralizing antibodies induced in a corresponding human subject administered a vaccine and/or immunogenic composition containing a Zika virus that is not adapted for non-human cell growth and/or comprises a different non-human cell adaptation mutation. In some embodiments, administration of a vaccine and/or immunogenic composition containing a non-human cell adapted Zika virus of the present disclosure induces generation of neutralizing antibodies to Zika virus in the human subject in an amount that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold greater than the than the amount of neutralizing antibodies induced in a corresponding human subject administered a vaccine and/or immunogenic composition containing a Zika virus that is not adapted for non-human cell growth and/or comprises a different non-human cell adaptation mutation. Methods of measuring neutralizing antibodies in a human subject are generally known to one of ordinary skill in the art.

Preferably, the therapeutically effective amount or immunogenic amount is sufficient to bring about treatment or prevention of disease symptoms. A suitable dosage is about 5 μg or about 10 μg, in particular about 10 μg,

The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting any aspect or scope of the present disclosure in any way.

EXAMPLES Example 1: Clonal Zika Virus Strain Generation

This example describes the production of Zika virus (ZIKAV) strains with a known research history.

Materials and Methods Vero Cell Maintenance

One vial of WHO Vero 10-87 cells was rapidly thawed in a water bath and directly inoculated into 19 mL pre-warmed DMEM (Dulbecco's modified minimal essential medium) containing penicillin-streptomycin, L-glutamine 40 mM, and 10% FBS in a T-75 cm² flask at 36° C.+/2° C., at 5% CO₂. Cells were allowed to grow to confluency and subcultured using Tryp1E. This flask was expanded to two T-185 cm² flasks, grown to confluency and subcultured to 31×T-185 cm² flasks and grown until the cells reached 100% confluency. Cells were harvested by trypsinization, centrifuged at 800×g for 10 minutes, and resuspended in DMEM containing 10% FBS and 10% DMSO at a concentration of 1.9×10⁷ cells/mL. One vial of the Vero cells was rapidly thawed and resuscitated as described above into a T-75 cm² flask. These were subcultured twice to produce a cell bank in 13×T-185 cm² flasks. After trypsinization, the cells were centrifuged at 800×g and resuspended in freezing media (DMEM containing 10% FBS, and 10% DMSO) at a concentration of 4.68×10⁵ cells/mL. This cell bank was aliquoted into cryovials.

The Vero cells were grown and maintained in DMEM containing penicillin-streptomycin, L-glutamine and 10% FBS (cDMEM-10%-FBS). Tryp1Express was used to maintain and trypsinize cells. Two days before viral adsorption, 6-well plates were seeded with 4-5×10⁵ cells/well in 3 mL of cDMEM-10%-FBS or 7×10⁵ cells in T-25 cm² flasks in 5 mL cDMEM-10%-FBS, or 1×10⁴ cells/well in 96-well plates in 0.1 mL cDMEM-10%-FBS. Incubators were monitored daily to maintain indicated temperatures. The Vero cell lines were stored in liquid nitrogen.

Plaque Assay

Viral titers were determined by plaque titration in freshly confluent monolayers of Vero cells grown in 6-well plates. Frozen aliquots were thawed and ten-fold dilution series of the aliquots were made in cDMEM-0%-FBS in 96-well plates. The diluted viruses were maintained on ice prior to inoculation of the Vero cell monolayers. At the time of assay, the growth medium was aspirated from the 6-well plate, and 100 μL of each virus dilution was added to the wells. Virus was adsorbed for 60 min at 36° C.±2° C., at 5% CO₂, with frequent (every 10 min) rocking of the plates to prevent drying of the cell sheets. Following viral adsorption, 4 mL of a first agarose overlay (1×cDMEM-2%-FBS+0.8% agarose) maintained at 40-41° C. was added to each well. The agarose was allowed to solidify for 30 min at room temperature, and the plates were then incubated upside down for 4-6 days at 36° C.+/2° C., at 5% CO₂. Two mL of a second agarose overlay containing 160 μg/mL of neutral red vital dye was added on day 4. Plaques were visualized on days 5 and 6.

Virus Quantification by TCID50 Assay

Viral titers were also determined by titration in freshly confluent monolayers of Vero cells grown in 96-well plates. Frozen aliquots were thawed and ten-fold dilution series of the aliquots were made in cDMEM-2%-FBS diluent in 96-well plates. The diluted viruses were maintained on ice prior to inoculation of the Vero cell monolayers. At the time of assay, the growth medium was aspirated from the 96-well plate, and 100 μL of each virus dilution was added to the wells. The plates were incubated for 5 days at 36° C.+/2° C., at 5% CO₂. The 50% Tissue Culture Infective Dose (TCID50) titer was calculated using the Reed/Muench calculator.

Test Articles

Zika virus strain PRVABC59 (one 0.5 mL vial on dry ice) was received from the Centers for Disease Control and Prevention (CDC) Zika virus identification was confirmed through RT-PCR. The strain tested negative for Alphavirus and mycoplasma contamination by PCR. This information is summarized in Table 1.

TABLE 1 PRVABC59 strain information Isolation Patient Strain Information information Prep info Analyses PFU PRVABC59 Human None Passage: Sequencing 6.7 log (Asian) serum; provided Vero(2)C6/36(1) by ion pfu/mL travel to Prep: Jan. 29, 2016 torrent: gene Puerto Rico Host: C6/36 accession in 2015 #KU501215 PFU by plaque assay Identity by RT-PCR (-) For alphaviruses by PCR (-) for mycoplasma by ATCC and ABM PCR

Sequencing

A QIAampViral RNA Mini Spin kit was used to extract RNA from stabilized virus harvests of each isolate according to manufacturer protocols. Extracted RNA from each isolate was used to create and amplify 6 cDNA fragments encompassing the entire Zika viral genome Amplified cDNA fragments were analyzed for size and purity on a 1% Agarose/TBE gel and subsequently gel purified using a Qiagen Quick Gel Extraction Kit. An ABI 3130XL Genetic Analyzer sequencer was used to conduct automatic sequencing reactions. Lasergene SeqMan software was used to analyze sequencing data.

Results

A ZIKAV strain with a known research history that was relevant to the current ZIKAV outbreak in the America's was sought. For this reason, ZIKAV strain PRVABC59 was chosen. To generate a well-characterized virus adapted for growth in Vero cells, the ZIKAV PRVABC59 was first amplified in Vero cells (P1).

Flasks of Vero cells (T-175 cm²), 100% confluent, were infected at an MOI of 0.01 in 4 mL of cDMEM-0%-FBS. Virus was adsorbed to the monolayer for 60 minutes at 36° C.±2° C., at 5% CO₂, then 20 mL of cDMEM-0%-FBS was applied for viral amplification at 36° C.±2° C., at 5% CO₂. The cell layer was monitored daily for cytopathic effect (CPE) following inoculation (FIG. 1). The supernatant was harvested after 96 hours by collecting the media and clarifying by centrifugation (600×g, 4° C., 10 min). The harvest was stabilized by adding trehalose to a final concentration of 18% w/v. The bulk was aliquoted into 0.5 mL cryovials and stored at −80° C.

The stabilized P1 harvest was analyzed for the presence of infectious virus on Vero cell monolayers by a TCID50 assay. Growth kinetics were monitored by taking daily aliquots beginning on hour 0. Peak titer was reached by hour 72 (FIG. 2).

P1 material was plaque-purified by titrating the harvest from day 3 on 6-well monolayers of Vero cells. Plaques were visualized on day 6, and 10 plaques to be isolated were identified by drawing a circle around a distinct and separate plaque on the bottom of the plastic plate. Plaques were picked by extracting the plug of agarose using a sterile wide bore pipette while scraping the bottom of the well and rinsing with cDMEM-10%-FBS. The agarose plug was added to 0.5 mL of cDMEM-10%-FBS, vortexed, labeled as PRVABC59 P2a-j and placed in an incubator overnight at 36° C.±2° C., at 5% CO₂.

Three plaques (PRVABC59 P2a-c) were carried forward for additional purification. Each isolate was plated neat in duplicate onto a fresh 6-well monolayer of Vero cells. This P2/P3 transition was plaque purified, and labeled PRVABC59 P3a-j.

Six plaques (PRVABC59 P3a-f) were carried forward for a final round of purification. Each isolate was plated neat in duplicate onto a fresh 6-well monolayer of Vero cells. This P3/P4 transition was plaque purified, and labeled PRVABC59 P4a-j.

Six plaques (PRVABC59 P4a-f) from the P4 plaque purification were blind passaged on monolayers of Vero cells in T-25 cm² flasks. Each plaque pick was diluted in 2 mL cDMEM-0%-FBS—1 mL was adsorbed for 1 hour at 36° C.±2° C., at 5% CO₂; the other 1 mL was stabilized with trehalose (18% v/v final) and stored at <−60° C. Following virus adsorption, cDMEM-0%-FBS was added to each flask and allowed to grow at 36° C.±2° C., at 5% CO₂ for 4 days. Virus supernatants were harvested, clarified by centrifugation (600×g, 4 C, 10 min), stabilized in 18% trehalose and aliquoted and stored at <−60° C. This P5 seed was tested by TCID50 for Zika virus potency (FIG. 3).

Confluent monolayers of T-175 cm² flasks of Vero cells were infected with each of the six clones of PRVABC59 (P5a-f) at an MOI of 0.01 in 4 mL cDMEM-0%-FBS. The virus was allowed to adsorb for 60 minutes at 36° C.+/2° C., at 5% CO₂, after which 20 mL of cDMEM-0%-FBS was added to each flask and allowed to grow at 36° C.+/2° C., at 5% CO₂. Vero cell monolayer health and CPE was monitored daily. Virus was harvested on days 3 and 5 as indicated (FIG. 4). The P6 strain harvests from days 3 and 5 were pooled, stabilized with 18% trehalose, aliquoted and stored <−60° C.

Each of the six clones of PRVABC59 (P6a-f) were tested for Zika virus in vitro potency (FIG. 5). The potency was determined by two different methods, TCID50 and plaque titration. The TCID50 was calculated by visual inspection of CPE (microscope) and by measuring the difference in absorbance (A560-A420) of the wells displaying CPE (yellow in color) compared with red (no CPE). The plates were read on a plate reader, and applied to the same calculator as the microscopically read-plates (absorbance). The values in TCID50 between the two scoring techniques are quite similar, while the values obtained by plaque titration are lower.

A summary of the generation of the P6 virus and characterization is shown in Table 2 below.

TABLE 2 Summary of virus passage and characterization for the generation of clonal ZIKAV strains Passage Seed production/purification Characterization P1 Virus amplification in Vero TCID50 titer P2 Amplify P1 by plaque titration plaque purification P3 Pick and passage plaques from P2 plaque plaque purification assay P4 Pick and passage plaques from P3 plaque plaque purification assay P5 Amplify P4 plaques (a-f) TCID50 titer P6 Amplify P5 (a-f) virus TCID50 titer, plaque phenotype, genotype, growth kinetics

An isolated Zika virus clone that closely resembled the envelope glycoprotein sequence of the original isolate was sought, since the envelope protein of flaviviruses is the dominant immunogenic portion of the virus. PRVABC59 clones P6a, P6c, P6d and P6f contained a G→T mutation at nucleotide 990 in the envelope region (G990T), resulting in an amino acid mutation of Val→Leu at envelope residue 330, whereas the envelope gene of PRVABC59 clones P6b and P6e were identical relative to the reference strain (GenBank ref KU501215.1) (Table 3 and FIG. 6).

TABLE 3 Sequencing of PRVABC59 P6 clones Envelope sequencing (reference gene from PRVABC59; accession #KU501215) Strain Nucleotide Amino Acid Mutation Comments PRVABC59 P6a Env-990: Env-330: Val/Leu Mutation in 3 of 4 G→T Val330→*Leu reads. PRVABC59 P6b Env-1404: Wild type Wild type Wild type relative to T→G silent reference. PRVABC59 P6c Env-990: Env-330: Val/Leu Mutation in 3 of 4 G→T Val330→*Leu reads. PRVABC59 P6d Env-990: Env-330: Val/Leu Mutation in 2 of 2 G→T Val330→*Leu reads. PRVABC59 P6e Wild type Wild type Wild type Wild type relative to reference. PRVABC59 P6f Env-990: Env-330: Val/Leu Mutation in 2 of 2 reads. G→T Val330→Leu 190 bp not sequenced (aa 421-484). PRVABC59 P6b Env-1404 Wild-type Silent Mutation in 2 of 2 reads T→G NS1-292 NS1-98 Trp/Gly Mutation in 2 of 2 reads T→G Trp98→*Gly PRVABC59 P6e NS1-292 NS1-98 Trp/Gly Mutation in 2 of 2 reads T→G Trp98→*Gly

The two clones lacking mutations in the envelope sequence were then subjected to full genome sequencing. Sequencing results are summarized in Table 3 above. Sequence analysis revealed a T→G substitution at nucleotide 292 in the NS1 region for both clones, resulting in a Trp→Gly mutation at NS1 residue 98. This mutation was also later confirmed through deep sequencing. The NS1 W98G mutation is located in the intertwined loop of the wing domain of ZIKAV NS1, which has been implicated in membrane association, interaction with envelope protein and potentially hexameric NS1 formation. While other tryptophan residues (W115, W118), are highly conserved across flaviviruses, W98 is not (FIG. 7). Interestingly, however, 100% conservation of the W98 residue is observed across 11 different ZIKAV strains, including those from the African and Asian lineages. The identified mutations in each strain are summarized in Table 4.

TABLE 4 Summary of mutations identified in PRVABC59 P6 clones Clone Nucleotide Amino Acid Mutations identified in envelope P6a G990T V330L P6b T1404G (silent) P6c G990T V330L P6d G990T V330L P6e none none P6f G990T V330L Additional mutations identified in genome P6b NS1-T292G NS1-W98G P6e NS1-T292G NS1-W98G Ref sequence: KU501215.1 (PRVABC59)

Phenotypic analysis of the ZIKAV PRVABC59 P6 stocks was conducted to characterize the ZIKAV clones. As illustrated in FIG. 8 and quantified in FIG. 9, each clonal isolate consisted of a relatively homogeneous population of large-sized plaques as compared to the P1 virus, which had a mixed population of large and small plaques. These data suggest the successful isolation of single ZIKAV clones.

Next, growth kinetics analyses in Vero cells of the ZIKAV PRVABC59 P6 clones were analyzed. Vero cells were infected with 0.01 TCID50/cell of each ZIKAV P6 clones in serum free growth medium. Viral supernatant samples were taken daily and simultaneously assayed for infectious titer by TCID50 assay. For all P6 clones, peak titer occurred between day 3 and 4 (˜9.0 log₁₀ TCID50/mL). There was no significant difference in growth kinetics of the various P6 clones (FIG. 10).

Taken together, the results indicate that a Zika virus seed was successfully generated. This seed selection required understanding of growth history, kinetics, yield, genotype, and phenotype of the virus. Importantly, clonal isolation of the Zika virus strains allowed for the successful purification of the virus away from contaminating agents (e.g., adventitious agents that may be in the parental human isolate). Interestingly, three sequential plaque purifications succeeded in quickly selecting Vero-cell adapted virus (strains P6a-f), where these strains were able to replicate well in serum-free Vero cell cultures, with strain P6a, c, d, and f harboring a mutation in the viral envelope protein, while strains p6b and p6e obtained a mutation in the viral NS1 protein (with no modification to the viral envelope). Additionally, the Vero-adapted strains enabled efficient and reproducible growth and manufacture of subsequent viral passages propagated from these strains. Without wishing to be bound by theory, the Env-V330L mutation observed in strains P6a, c, d, and f may potentially be a result of in vitro adaptation, as a mutation at Env 330 was also observed upon passaging in Vero cells (Weger-Lucarelli et al. 2017. Journal of Virology). Because the envelope protein is the dominant immunogenic epitope of Zika virus, strains containing a Vero adaptive mutation in Env may negatively impact vaccine immunogenicity. Without wishing to be bound by theory, the adaptation mutation in protein NS1 appears not only to enhance viral replication, but may also reduce or otherwise inhibit the occurrence of undesirable mutations, such as in the envelope protein E (Env) of the Zika virus. In addition, NS1 may be known to bind to the Envelope protein during the life cycle of the virus. This mutation (NS1 W98G) may be implicated in changing the ability of the NS1 to associate, and possibly co-purify, with the virus during downstream processing. NS1 is also known to be immunogenic, and could be implicated in the immune response to the vaccine.

Example 2: Preclinical Immunogenicity and Efficacy of a Purified Inactivated Zika Virus Vaccine (PIZV) Derived from the P6b and P6e Strains

The following example describes the preclinical immunogenicity and efficacy in CD1 and AG129 mice of an inactivated Zika virus vaccine (PIZV) derived from the P6b and P6e strains. As described in Example 1, six clones were generated from the epidemically relevant PRVABC59 strain, and two (P6b and P6e) were chosen for further preclinical immunogenicity and efficacy studies.

Materials and Methods Purification, Inactivation and Formulation of a Zika Virus Vaccine

A lot of inactivated ZIKAV vaccine, suitable for use in preclinical immunogenicity and efficacy studies, was generated and characterized. Virus was amplified from the P6b and P6e strains by infecting flasks of confluent Vero cells at a MOI of 0.01. Virus was adsorbed for 1 hour at 36° C.±2° C./5% CO₂. Following adsorption, 20 mL of cDMEM-0%-FBS was added to each flask, and incubated at 36° C.±2° C./5% CO₂ for five days. Cell supernatants were harvested on day 3 and 5 post-infection, and cell debris was clarified by centrifugation.

For each isolate, clarified supernatants were pooled, stabilized in DMEM containing 18% trehalose and stored at <−60° C. Pooled, clarified virus supernatants were thawed in a 37° C. water bath and treated with benzonase overnight at 4° C. Following benzonase treatment, each sample was applied to a Sartorius PP3 depth filter. Following depth filtration, each sample was applied to a Centricon Plus-70 tangential flow filtration (TFF) device. Retentate was buffer exchanged, diluted, and applied to a Sartorius SartobindQ IEXNano. Each sample was applied to a second Sartorius SartobindQ IEXNano and eluted using a 3 step-elution process with 250 mM, 500 mM, and 750 mM NaCl. Following MonoQ chromatography and dilution, each 250 mM eluate was applied to a Centricon Plus-70 cross flow filtration (CFF) device for buffer exchange, diluted to 35 mL with PBS, and stored at 2-8° C.

For formalin inactivation, freshly prepared 1% formaldehyde was added dropwise to each purified sample with gentle swirling to obtain a final formaldehyde concentration of 0.02%. Samples were incubated at room temperature (˜22° C.) for 14 days with daily inversion. Formaldehyde was neutralized with sodium metabisulfite for 15′ at room temperature before being applied to a Centricon Plus-70 tangential flow filtration (TFF) device. Buffer exchange was performed four times by the addition of 50 mL Drug Substance Buffer (10 mM NaH₂PO₄, 50 mM NaCl, 6% sucrose, pH 7.4). Each sample was then diluted to 15 mL with Drug Substance Buffer, sterilized using a 0.2 m syringe filter, aliquoted into sterile stoppered glass vials (0.5 mL per vial) and frozen at <−60° C.

Virus inactivation was confirmed by TCID50 assay and double infectivity assay. Briefly drug substance sample was applied to C6/36 cells and allowed to amplify for 6 days. Supernatant from C6/36 cells was applied to Vero cells and CPE was monitored for 8 days. For drug product formulation, vials of PIZV drug substance were thawed, pooled according to sample type, and diluted to 1 μg/mL or 10 μg/mL in PBS with or without Alhydrogel (Brenntag; 0.5 mg/mL final, 0.050 mg/dose) and incubated overnight at 2-8° C. with gentle agitation. The resulting drug product lots were then aliquoted into sterile stoppered glass vials and stored at 2-8° C. until use. FIG. 11 provides a summary of the steps used to prepare drug product.

Mouse Immunization and Challenge

For the immunogenicity study, six-week old male and female Swiss-ICR (CD-1) mice were divided into 6 groups (n=10/group). On Day 0, mice in groups 1-5 were inoculated with 0.1 mL of vaccine by the intramuscular (i.m.) route (2×0.05 mL injections). Mice in group 6 were inoculated with PBS as a placebo control. Mice were boosted on day 28 and 56 using the same dosage and vaccine type as day 0. Blood samples were collected on day −1 (pre-immune), day 27 (prime), day 42 (boost 1) and day 70 (boost 2).

For the immunogenicity and efficacy study, four-week old male and female AG129 mice were divided into 7 groups (n=5/group). On Day 0, mice in groups 1-6 were inoculated with 0.1 mL of vaccine by the intramuscular (i.m.) route (2×0.05 mL injections). Mice in group 7 were inoculated with PBS as a placebo control. Mice were boosted on day 28 using the same dosage and vaccine type as on day 0. Blood samples were collected from the tail vein on day −1 (pre-immune), day 27 (prime) and day 55 (boost). At the time of euthanization, mice were bled via cardiac puncture under deep anesthesia with isofluorane (terminal). On day 56, mice were intraperitoneally challenged with 10⁴ plaque forming units (PFU) of ZIKAV PRVABC59.

Serum Transfer

Serum was collected from PIZV-vaccinated and challenged AG129 mice, and were frozen after pooling (groups 1, 2, 4, and 5 of Table 6). The serum pool was thawed, and the test articles were generated by three-fold dilutions of the serum pool in PBS. A placebo was generated using 3-fold dilutions of AG129 normal mouse serum in PBS.

The test articles were administered as 0.1 mL intraperitoneal injections into AG129 mice (an equivalent volume of the placebo article was administered to control mice). Animals were then challenged intraperitoneally with 10⁴ plaque forming units of Zika virus strain PRVABC59 in 100 μL.

Allowable blood volume by weight was collected as whole blood by tail bleeding from ten mice on day −11 (pre-immunization). Whole blood was collected from each mouse on day 1 (primary, circulating Nab) and day 4 (viremia) by tail bleeding. Terminal bleeding after lethal challenge was performed by heart puncture under deep anesthesia for larger volume before euthanization by cervical dislocation. Blood samples were collected in microtainer SST serum separation gel tubes and allowed to clot for at least 30 min before separation of serum by centrifugation (10,000×g for 2 min) and frozen at −80° C.

Plaque Reduction Neutralization Test

Neutralizing antibody titers were determined by a plaque reduction neutralization test (PRNT) as described previously (See e.g., Osorio et al. Lancet Infect Dis. 2014 September; 14(9):830-8).

Reporter Virus Particle (RVP) Neutralization Assay

Neutralizing antibody titers were analyzed by titration of serum samples with a constant amount of Zika RVPs in Vero cells grown in 96-well plates. RVPs contained the prME proteins of Zika (strain SPH2012) and a Dengue-based Renilla luciferase reporter. Briefly, sera were heat inactivated at 56° C. for 30 min, diluted, and then incubated at 37° C. with RVPs. The serum/RVP mixture was then mixed with Vero cells and incubated for 72 hours at 37° C.±2° C./5% CO₂ before detection with luciferase substrate. Data was analyzed using JMP11 non-linear 4 parameter analysis, normalized to a positive tracking control and effective dose 50% (EC50) was reported.

Unless indicated to the contrary, all additional experimental methods were carried out as described in Example 1 above.

Results

To assess the immunogenicity of the PIZV candidates in 6 week old male and female CD-1 mice, groups of CD-1 mice (N=10/group) were immunized by the i.m. route with either a 0.1 μg (+alum), 1.0 μg (+alum) dose of a vaccine derived from either ZIKAV PRVABC69 P6b or P6e virus strains. To assess the need for adjuvant, a group of animals was vaccinated with 0.1 μg of vaccine derived from P6e and lacking alum adjuvant. Vaccinations occurred on days 0, 28, and 56, with group 6 receiving PBS as a placebo control (FIG. 12A and Table 5).

TABLE 5 PIZV formulations and challenges in CD-1 mice Group Strain Dose (μg) Alum (μg) N 1 P6b 0.1 0.50 10 2 P6b 1.0 0.50 10 3 P6e 0.1 0.50 10 4 P6e 1.0 0.50 10 5 P6e 0.1 — 10 6 Placebo (PBS) — — 10

Following vaccination, serum samples collected after primary (day 27), secondary (day 40) and tertiary (day 70) immunizations were tested for ZIKAV-specific neutralizing antibodies by RVP neutralization assay (FIG. 12B). Twenty-seven days after receiving the first dose, a slight neutralizing antibody response was observed in mice vaccinated with PIZV derived from either clone containing alum, as compared to the PBS placebo control group. Importantly, this response increased significantly upon a second immunization (day 40), but was not additionally enhanced upon immunization with a third dose (day 70). No neutralizing antibody response was observed in mice vaccinated with non-adjuvanted vaccine (FIG. 12B).

To assess the immunogenicity and protective efficacy of the PIZV candidates, groups of 4 week old AG129 mice (n=5/group) were immunized by the i.m. route with either a 0.1 μg dose (+alum), 1.0 μg dose (+alum) or 0.1 μg dose (−alum) of a vaccine derived from either the ZIKAV PRVABC59 P6b or P6e stocks on days 1 and 28 (FIG. 13A and Table 6).

TABLE 6 PIZV formulations and challenges in AG129 mice Group Sex Strain Dose (μg) Alum (μg) N 1 F P6b 0.1 0.50 5 2 F P6b 1.0 0.50 5 3 F P6b 0.1 — 5 4 M P6e 0.1 0.50 5 5 M P6e 1.0 0.50 5 6 M P6e 0.1 — 5 7 M Placebo (PBS) — — 5

Following vaccination, vaccinated and control mice were intraperitoneally challenged at day 56 with 10⁴ PFU of ZIKAV PRVABC59 (low passage). Serum samples collected after primary (D27) and secondary (D55) immunizations were tested for ZIKAV-specific neutralizing antibody response (FIG. 13B and Table 7). Only groups receiving the high dose of alum-adjuvanted vaccine (groups 2 and 5) elicited a neutralizing antibody response after a single immunization, which increased dramatically after boosting. In contrast, groups receiving either the low or high dose of alum-adjuvanted vaccine produced a high neutralizing antibody response after a second dose. Upon receiving two doses of vaccine, there was no statistical difference between groups of mice receiving alum-adjuvanted vaccine, regardless of the dosage or the derivation from the P6 clone.

TABLE 7 ZIKAV-specific neutralizing antibody response Serum neutralizing antibody titers D27 (prime) D55 (boost) Group Formulation GMT % sc GMT % sc 1 P6b 0.1 μg + <20 40 1280 100 alum 2 P6b 1.0 μg + 135 80 2229 100 alum 3 P6b 0.1 μg − <20 0 <20 0 alum 4 P6e 0.1 μg + <20 20 640 100 alum 5 P6e 1.0 μg + 30 100 905 100 alum 6 P6e 0.1 μg − <20 0 <20 20 alum 7 PBS <20 0 <20 0

All groups were also monitored for mortality, morbidity and weight loss for 21 days post challenge. Viremia following challenge was detected and quantitated by plaque titration. Mice vaccinated with a low or high dose of PIZV candidates formulated with alum (groups 1, 2, 4 and 5) were fully protected from lethal ZIKAV challenge, as assessed by the plaque reduction neutralization test (PRNT) assay, as well as a comparable secondary neutralization assay (Table 8). No weight loss or clinical signs of illness were observed in vaccinated mice, none had detectable infectious viremia three days post challenge, and all mice vaccinated with either low or high dose antigen+alum adjuvant survived to 21 days post-challenge (FIGS. 14-16). In contrast, challenge of all naïve mice resulted in high viremia on day 2 post challenge and morbidity/mortality between day 10 and 18 post challenge (median survival=D13). Additionally, challenge of mice vaccinated with a non-alum-adjuvanted low dose vaccine derived from strain P6b resulted in high viremia on day 2 post challenge and a median survival day similar to the placebo control group, while mice vaccinated with a non-alum-adjuvanted low dose derived from clone e remained partially protected with a median survival of 19 days. These results indicate immunization is more effective with alum, secondary immunization may be a requirement, and that low dose was as effective as high dose.

TABLE 8 Serum neutralizing antibody titers Serum neutralizing antibody titers Terminal (post challenge) Pool PRNT₅₀ Secondary assay Alum (1, 2, 4, 5) 10240 20480 No alum (3, 6) 2560 2560 PBS (7) 1280 1280

Additionally, the presence of NS1 in the vaccine drug substance (DS) produced from whole inactivated P7b and P7e virus (one additional passage from the P6b and P6e strains, respectively) was tested. A sandwich ELISA was performed using plates pre-coated with a monoclonal antibody reactive to both Asian and African lineages of Zika virus NS1, but non-cross-reactive to Dengue NS1. Duplicate 2-, 4-, 8-, 16-, and 32-fold dilutions of DS were prepared, and were compared to a standard curve using recombinant purified NS1 in duplicate at a concentration of 0-8 ng/mL. Duplicate dilutions of DS buffer alone were prepared as negative controls. Bound NS1 was detected with anti-NS1 HRP-conjugate, and absorbance (A450-A630) of the wells with DS buffer alone was subtracted from the absorbance measured in the wells containing the matching DS samples. Results of the sandwich ELISA are shown in Table 9 below. Interestingly, NS1 was observed to co-purify with the vaccine drug substance preparations, suggesting that viral NS1 may be an immunogenic component of the whole inactivated virus vaccine.

TABLE 9 NS1 ELISA Strain in Predicted Predicted vaccine Sample log Std Lower Upper Dilution concentration preparation OD ng/mL Error 95% 95% Factor (ng/mL) P7b 3.61 0.951 0.018 0.915 0.986 32 ~285 P7e 3.79 0.980 0.023 0.935 1.024 32 ~306

The threshold of neutralizing antibody (Nab) needed to confer protection from wild-type Zika virus challenge after passive transfer of antibodies was next tested. (Tables 10A and B).

TABLE 10A design of passive transfer study in AG129 mice Group Test Article Serum dilution Predicted Nab titer before IP 1 100 μL 1/3 6827/3.83 2 100 μL 1/9 2276/3.36 3 100 μL  1/27  759/2.88 4 100 μL  1/81  253/2.40 5 100 μL  1/243  84/1.93 6 100 μL  1/729  28/1.45 7 100 μL   1/2187   9/0.97 8 100 μL PBS —

TABLE 10B Timing of passive transfer study in AG129 mice Description Study Day Passive transfer Day 0 Primary Bleed Day 1 (AM) Challenge (PM) Day 1 Viremia Bleed Day 4 Terminal Bleed Day 29 for survivors

Pooled serum from vaccinated and challenged AG129 mice was serially diluted 3-fold in PBS and intraperitoneally injected into 7 groups (N=5/group) of 5-6 week old AG129 mice. Pre-immune AG129 mouse serum was used as placebo control (group 8). Following passive transfer (˜16-19 hours later), whole blood was collected and serum was separated by centrifugation from each mouse prior to virus challenge for determination of circulating neutralizing antibody titer (FIG. 17). Just prior to virus challenge, groups of mice (designated groups 1, 2, 3, 4, 5, 6, 7, 8) had mean log₁₀ neutralizing antibody titers of 2.69, 2.26, 1.72, 1.30, <1.30, <1.30, <1.30, <1.30, respectively.

Twenty four hours following passive transfer of ZIKV nAbs, mice were intraperitoneally challenged with 10⁴ pfu of ZIKV PRVABC59. Following challenge, animals were weighed daily and monitored 1-3 times a day for 28 days for signs of illness. A clinical score was given to each animal based on the symptoms (Table 11). Animals that were moribund and/or showed clear neurological signs (clinical score ≥2) were humanely euthanized and counted as non-survivors.

TABLE 11 Description of clinical scores given while monitoring for morbidity and mortality Score Description 0 Normal appearance and behavior 1 Slightly ruffled fur and/or general loss of condition 2 Increases in above behavior/appearance, breathing changes, twitching, anti-social behavior 3 First signs of neuropathy-Severely hunched posture, partial paralysis (immobility, unsteady gait, flaccid hind legs, severe twitching), or full paralysis 4 Found dead without showing signs of score of 2 or 3 first

Signs of disease began appearing nine days after challenge in the control group (group 8) and groups 5-7, with a corresponding loss in weight (FIG. 18). Whole blood was collected and serum was separated by centrifugation from each animal three days post challenge. Serum samples were analyzed for the presence of infectious ZIKV using a plaque titration assay (FIG. 19). The mean infectious titer (log 10 pfu/mL) for mice in groups 1-8 were: 1.66, 2.74, 4.70, 4.92, 7.24, 7.54, 7.54 and 7.46, respectively. Importantly, mice in groups 1-4 with detectable levels of ZIKV neutralizing antibodies (≥1.30 log₁₀) had statistically significant lower levels (102.5- to 106.0-fold lower titers) of viremia (p=0.0001, 0.0003, 0.0007 and 0.0374) than control mice. These results suggested that detectable levels of ZIKV neutralizing antibodies (≥1.30 log₁₀) reduced viremia in a dose-dependent manner

The median survival day of mice in groups 1-8 were: not determined, day 17, day 17, day 13, day 11, day 11, day 11, and day 10, respectively (FIG. 20). Importantly, the survival curves for groups of mice with detectable ZIKV neutralizing antibody titers (groups 1-4) were statistically different compared to the control group (group 8) (p=0.0019, 0.0019, 0.0019, 0.0153, respectively). These results suggested that detectable levels (≥1.30 log₁₀) of ZIKV neutralizing antibodies delayed onset of disease in a dose-dependent manner

Finally, the ZIKV neutralizing antibody titer of each animal was graphed against its corresponding viremia titer and linear regression analysis was performed. A highly inversely correlated relationship between ZIKV neutralizing antibody titers and viremia levels at day 3 post-challenge was observed (FIG. 21). A summary of the results from the passive transfer studies is shown in Table 12 below.

TABLE 12 Summary of passive transfer results Circulating Viremia ZIKV (D3) % Median Serum nAb log10 survival survival Group dilution GMT pfu/mL (D28) day 1 1/3 2.69 ± 0.17 1.66 ± 0.62 20 24 2 1/9 2.26 ± 0.13 2.73 ± 0.68 0 17 3 127 1.72 ± 0.16 4.69 ± 0.77 0 17 4 1/81 1.30 ± 0.16 4.94 ± 1.29 0 13 5 1/243 <1.30 7.25 ± 0.10 0 11 6 1/729 <1.30 7.54 ± 0.31 0 11 7 1/2187 <1.30 7.52 ± 0.39 0 11 8 PBS <1.30 7.47 ± 0.37 0 10

While no groups of mice receiving ZIKAV neutralizing antibodies were fully protected from lethal ZIKAV challenge in this experiment, reduced viremia levels and delayed onset of disease in a dose-dependent manner among the groups of mice with detectable levels of circulating ZIKAV neutralizing antibody titers was demonstrated.

Taken together, preclinical data from both CD-1 and AG129 mouse studies indicate that a PIZV derived from separate and well-characterized viral clones are immunogenic and able to provide protection against challenge with wild-type ZIKAV. Importantly, a low and high vaccine dose elicited a similar neutralizing antibody response after two doses, and provided similar levels of protection against lethal ZIKAV challenge. Interestingly, mice vaccinated with an unadjuvanted PIZV candidate also showed partial protection from ZIKAV challenge. Vaccine antisera significantly diminished viremia in passively immunized AG129 mice, and prolonged survival against lethal ZIKAV challenge. These results also demonstrate that the well-characterized PIZV candidates were highly efficacious against ZIKAV infection in the highly ZIKAV-susceptible AG129 mouse model.

Additionally, it was found that the sequence of a PRVABC59 (from PRVABC59 P6e) at passage 7 was genetically identical to that of passage 6. This was surprising given that flaviviruses are generally regarded as genetically labile. PRVABC59 P6e was selected as the master virus seed due in part to its genetic stability over 7 passages. Without wishing to be bound by theory, it is believed that this enhanced genetic stability may be due to the single amino acid substitution (W98G) in the wing domain of NS1, as this was the only mutation observed in the Vero cell-adapted PRVABC59 P6 genome. Additionally, genetic stability and homogeneity is advantageous in that it reduces variability and increases reproducible production of subsequent strains that may be used for vaccine formulation.

Example 3: Preclinical Assessment of the Phenotype of the P6a and P6e Strains Materials and Methods

AG129 mice (lacking interferon α/β and γ receptors) are susceptible to ZIKV infection and disease, including severe pathologies in the brain. 14-week-old AG129 mice were intraperitoneally infected with 10⁴ and 10³ pfu of the ZIKV passage 6 clones a (P6a) and e (P6e).

Mice were weighed and monitored daily (up to 28 days) for clinical signs of illness (weight loss, ruffled fur, hunched posture, lethargy, limb weakness, partial/full paralysis). Additionally, analysis of viremia was performed by plaque titration of serum samples collected three days post-challenge as described in Example 1.

Results

Infection with P6e resulted in 100% mortality (median survival time=12.5 days), while infection with P6a resulted in only 33% mortality (median survival time=undetermined) (FIG. 22). In agreement with this, preMVS P6e infected mice showed greater weight loss as compared to PRVABC59 P6a infected mice (3). No statistical difference was found in mean group viremia levels between groups of mice infected with PRVABC59 P6a or P6e (FIG. 24). These data suggest that growth kinetics alone may not be a key determinant (since both strains produced similar viremia, and similar peak titers in vitro) and that a characteristic of the Envelope protein could be important for virulence (of a wildtype strain) and immunogenicity (of an inactivated candidate).

Example 4: Completeness of Inactivation Assay to Determine Effectiveness of Inactivation

A double-infectivity assay also called completeness of inactivation (COI) assay was developed to determine the effectiveness of formalin-inactivation (0.01% formaldehyde) and potential residual infectious viral activity of purified inactivated zika virus (PIZV) bulk drug substance (BDS).

Sample preparation: Four Purified Inactivated Zika Vaccine (PIZV) lots (Tox lots 1-4) of clone e as described above were manufactured by growth in Vero cells. Supernatants from 4 daily harvests (totaling about 4000 mL) were purified by chromatography followed by addition of formaldehyde to a final concentration of 0.01%. w/v Inactivation was allowed to proceed for 10 days at 22° C. In Process Control (IPC) samples were removed on a daily basis from the bulk drug substance (BDS) during inactivation for characterization and analytics. The daily IPC samples were neutralized with sodium metabisulfite and dialysed into DMEM (viral growth media). The samples contain the purified inactivated Zika virus. On the final day of inactivation, the remaining volume of BDS samples was not neutralized, but was processed with TFF to remove formaldehyde and buffer exchanged into PBS.

Completeness of inactivation assay (COI): The COI assay was used for analysis of the effectiveness of inactivation in the daily IPC samples to understand the kinetics of inactivation, and the final BDS. For maximum sensitivity, two cell lines, Vero and C6/36, were initially utilized in this assay to detect potential live virus in the IPC and DS samples. When Zika virus infects Vero cells in the presence of growth medium containing phenol red, the by-products of cell death cause a drop in pH. Consequently, the media color changes from red/pink to yellow, indicative of this acidic shift in the media pH. This phenomenon is caused by the apoptosis and cytopathic effects (CPE), which refers to the observed changes in the cell structure of host cells that are caused by viral invasion, infection, and budding from the cells during viral replication. Ultimately, while both C6/36 mosquito and Vero cells are a permissive cell line for infection, Zika virus infection kills only Vero cells in vitro. Therefore, Vero cells were used as the indicator cell line for the assay. In contrast, C6/36 cells which are derived from a natural host vector for Zika virus do not exhibit a CPE upon Zika infection and do not lyse. The media does not change color and the viability of the C6/36 cells is not altered.

The assay is thus split in two parts: The first part of the assay allows for parallel amplification of potentially live viral particles on 96-well plates of the two susceptible cell lines for six days. The second step of the assay involves the transfer of the supernatant of the 96-well plates (including potentially amplified particles) onto 6-well plates containing monolayers of Vero cells, and incubation for another 8 days to allow for viral infection and a cytopathic effect to develop on the Vero cells. Any CPE observed was confirmed using a light microscope.

Although described in detail with respect to the use of 96 well plates in the first part of the assay, i.e. the culture in C6/36 cells, and six well plates in the second part of the assay, i.e. the culture of Vero cells to observe a cytopathic effect, the assay can be easily scaled up according to the following table:

Assay part 2: transfer to Vero (must accommodate Assay part 1: BDS application (must fall within pooled recommended vol range) volume for # vessels # vessels transfer) vol required required pooled vol Recommended inoculum for 15X for 15X volume transferred Surface volume mL per well scale-up; scale-up; for mL inoculum plate or area range (for sample (or per 2-fold 5-fold transfer sample per well flask (cm²) growth) per cm² flask) dilution dilution (mL) per cm² (or flask) 96-well 0.32 100-200 uL 0.3125 0.1 format 12-well 3.8 0.076-1.14 ml 0.3125 1.188 6.48 16.21 11.88 format 6-well 9.5 1.9-2.9 mL 0.3125 2.969 4.32 10.81 17.81 0.0526 0.1 format T25 flask 25 5-7.5 mL 0.3125 7.813 9.86 24.64 7.813 0.0526 1.32 format T75 flask 75 15-22.5 mL 0.3125 23.438 3.29 8.21 23.438 0.0526 3.95 format T150 flask 150 30-45 mL 0.3125 46.875 1.64 4.11 46.88 0.0526 7.89 format T175 flask 175   35-52.5 0.3125 54.688 1.41 3.52 54.69 0.0526 9.21 format T235 flask 235   47-70.5 0.3125 73.438 1.05 2.62 73.44 0.0526 12.36 format T300 flask 300 30-40 mL? 0.3125 93.750 0.82 2.05 93.75 0.0526 15.78 format CF1 6/36 150-200 0.3125 198.750 0.39 0.0526 33.45 CF2 1272 300-400 0.3125 397.500 0.19 0.0526 66.91 CF10 63360 1500-2000 0.3125 19800.000 0.00 0.0526 3332.74 It is apparent that during the scale up the volume of sample per cm² of vessel remains constant for part 1 and the same viral infection conditions are kept in part 2.

COI assay control: The titer and back titration controls for this assay were performed using Vero indicator cells and scored in a TCID50 96-well format with wells scored positive based on the media color change from pink to yellow, as a surrogate for cell death, or the presence of CPE.

Virus titer control test: Two independent replicates of the control virus (PRVABC59) of known titer were subjected to a 10-fold dilution series in media containing 2% PBS, and 100 μL of each dilution was added to four wells of a 96-well plate containing Vero cells. Plates were incubated for 5 days, then wells containing CPE were recorded and virus titer was calculated using the Reed-Meunch calculator.

Virus back titration control test: The control virus of known titer was serially diluted to 200 TCID50. Two independent replicates of the 200 TCID50 control virus were subjected to a 2-fold dilution series in media containing 2% FBS, and 100 μL of each dilution was added to four wells of a 96-well plate containing Vero cells. Cells were incubated for 5 days, then wells containing CPE were recorded and virus titer was calculated using the Reed-Meunch calculator.

Detailed COI Protocol:

-   -   1. First part of the assay: Vero (1.4E⁺⁰⁵ cells/mL) and Aedes         aegypti mosquito C6/36 (4E⁺⁰⁵ cells/mL) cells were seeded in         96-well plates two days prior to addition of the samples. The         Vero cells were cultured in DMEM+10% final FBS+2% L-glutamine+1%         penicillin/streptomycin at 37° C. C6/36 cells were cultured in         DMEM+10% FBS+2% L-glutamine+1% Penicillin/streptomycin+1%         nonessential amino acids at 28° C.     -   2. Three independent replicates of the 200 TCID50 control virus         (prepared in the virus back titration control test) or the DS         samples were diluted (5-fold and 10-fold dilutions) into media         containing 2% FBS.     -   3. The cells in 96-well plates were inoculated with the samples.         Prior to the infection of the cell monolayers in the 96-well         plates, the sample was vortexed to disrupt any possible         aggregation. 100 μL of each dilution was applied to each of 5         wells into two separate 96-well plates containing Vero and C6/36         cells, respectively.     -   4. Media alone was included in another well for each cell type         as a negative CPE control.     -   5. Plates were incubated for 6 days at the appropriate         temperature for the cell line.     -   6. Second part of the assay: To allow live virus to be further         amplified and visualized by CPE on a permissive cell line, the         entire volume of each 96-well supernatant from both Vero and         C6/36 cells was transferred to individual wells of 6-well plates         of Vero cells. Inoculation proceeded for 90 minutes with rocking         at 15 minutes intervals.     -   7. Medium containing 2% FBS was added to the wells and plates         were incubated for an additional 8 days for subsequent detection         of the amplified samples as a function of CPE. The inactivation         was considered to be incomplete if any of the replicates of the         DS showed CPE at the end of day 8.     -   7. The presence of live/replicating virions was visualized by         the formation of plaques or CPE on susceptible cell monolayers         after transfer to the 6-well plate, and incubation for 8 days to         allow for viral replication. The % CPE scoring in the 6-well         plates at the end of the assay was calculated as follows:         -   Each 6-well plate of Vero cells was examined for CPE by             visualization of colorimetric change, followed by             confirmation of CPE by inspection under an inverted light             microscope.         -   Each 6-well plate represented one of the replicates of the             DS dilutions prepared in the 5 and 10-fold dilutions             described above (5 wells, plus one well containing media             alone).     -    Therefore, % CPE for each replicate reflected the number of         wells with CPE out of 5 total wells per sample (120 total wells         are used per assay). Mean % CPE and standard deviation were         calculated based on three replicates of each dilution.

Results: The daily samples were analyzed in each of the Tox lots #1-4 as shown in the following tables.

TABLE A Kinetics of Inactivation, Tox lot #1 Mean Sample Transfer % CPE STDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10 Day 1 Vero-to-Vero 0 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3 Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 0 1:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10 Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1 C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1 C6/36-to-Vero 6.7 12 1:10 Day 2 C6/36-to-Vero 13.3 12 1:10 Day 3 C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero 0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day 10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE B Kinetics of Inactivation, Tox lot #2 Mean Sample Transfer % CPE STDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10 Day 1 Vero-to-Vero 100 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3 Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 0 1:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10 Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1 C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1 C6/36-to-Vero 100 12 1:10 Day 2 C6/36-to-Vero 13.3 12 1:10 Day 3 C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero 0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day 10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE C Kinetics of Inactivation, Tox lot #3 Mean Sample Transfer % CPE STDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10 Day 1 Vero-to-Vero 27 12 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3 Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 0 1:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10 Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1 C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1 C6/36-to-Vero 87 12 1:10 Day 2 C6/36-to-Vero 27 12 1:10 Day 3 C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero 0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day 10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE D Kinetics of Inactivation, Tox lot #4 Mean Sample Transfer % CPE STDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 93 12 1:10 Day 1 Vero-to-Vero 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3 Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 0 1:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10 Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1 C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1 C6/36-to-Vero 33 23 1:10 Day 2 C6/36-to-Vero 7 12 1:10 Day 3 C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero 0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day 10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

Compiled kinetics of inactivation data: COI data for samples from the four toxicology lots were compared to infectious potency (TCID50) determined as described above and to RNA copy. The RNA copy was determined by purifying nucleic acids from the sample and amplifying Zika RNA with serotype-specific primers using an RT-PCR kit. The result shown in FIG. 25 shows that the sensitivity of the COI assay is significantly greater than that of TCID50.

Performance characteristics of the COI assay—Accuracy: The target dilutions (TCID50/well) and their respective proportions of CPE were used to determine relative accuracy. For the Vero cells, there was a statistically significant linear relationship between the observed and expected proportions of positive CPE. The slope of the line relating observed and expected results is 0.92 with a 95% confidence interval (CI) of 0.83 to 1.01 that overlaps 1 indicate 100% accuracy. For the C6/36 cells, there is a statistically significant linear relationship between the observed and expected proportions of positive CPE. The slope of the line relating observed and expected results is 0.88 with a 95% confidence interval (CI) of 0.80 to 0.95 indicate that a slight bias (5-20%) was seen with this cell line. Both cell lines demonstrate satisfactory accuracy (relative).

Performance characteristics of the COI assay—Limit of Detection (LoD): The sensitivity of the assay was assessed for both the C6/36-to-Vero and Vero-to-Vero plates. As described above, the data was fitted using least squares regression of the proportion of +ve CPE observed per total wells plated with titer dilutions plated starting at 10.00 TCID50/well down to a lower titer of 0.08 TCID50. Furthermore, negative controls (0.00 TCID50/well) were included for each dilution within the plates. CPE scoring was performed for each dilution across both the C6/36-to-Vero and Vero-to-Vero plates. A clear relationship between the CPE and log input virus titer was seen. This displays the logistic (sigmoidal) relationship between the proportion of CPE positive wells relative to the log₁₀ concentration of TCID50/well together with a lower and upper 99% confidence limit. At a −2 log₁₀ concentration (=0.01 TCID50/well), a model based on and accounting for all fixed and random sources variation in the qualification data predicted 0.85%, or 0.01 when rounded up at 0.01 TCID50/well, with a lower 99% confidence limit of 0.42%. Since the lower 99% confidence limit does not include zero, there is a very small quantifiable (<1%) chance the 0.85% CPE wells could have arisen from 0 TCID50/well (i.e., due to noise). This establishes a detection limit for the assay of at least 0.01 TCID50/well (i.e., the lowest amount of live Zika particles in the sample which can be detected). That is, when rounded up, 1 in 60 wells will be CPE positive or given these parameters, the lowest theoretical proportion of the CPE+ve that could be detected in 60 wells would be 1.67%, or 0.0167.

The cell types (C363 and Vero) were compared for relative sensitivity, with the C6/36 demonstrating that a lower dilution of virus titer could be detected compared to Vero cells as shown in FIG. 26; at the same virus input level (0.31 TCID50), the proportion of CPE positive wells is higher for C6/36 relative to Vero cells.

The lowest virus input value used during the qualification of this assay was 0.02 TCID50 (−1.61 log TCID50). Using the fitted curve for C6/36 cells, this results in 0.035 or 3.5% of the wells scoring CPE positive (1 in 28 wells). If the curve is extrapolated towards the lowest practical level of 0.167 or 1.6%, then this equates to a virus input level of 0.015 TCID50 (−1.82 log TCID50). However, the impact of transmitted assay variance needs to be considered when determining the lowest levels of infectious virus that can be detected as reflected in the +ve CPE results. This noise arises from generation of the working stock of input virus. Comparison of the target TCID50 and the back-titration calculation shows the TCID50 of the working stock virus exhibited a standard deviation (SD) of 85 TCID50/mL, derived from a mean of 213 when targeting a stock TCID50/mL concentration of 200. The % CV calculates to ˜40% with a bias of about +7%. This noise was factored into the logistic regression model to generate confidence intervals around the targeted values for the virus dilutions. At a target value of 0.01 TCID50/well, a model based on and accounting for all fixed and random sources of variation in the qualification data across the two sites predicts 0.86% of wells will be CPE positive (1 in 60 wells). Since the lower 99% confidence limit does not include zero, there is a very small quantifiable (<1%) chance the 0.85% CPE-positive wells could have arisen from 0 TCID50/well due to noise (FIG. 27). This establishes a detection limit for the assay: 0.01 TCID50/well is the lowest amount of live Zika particles in the sample which can be detected.

Performance characteristics of the COI assay—Range: The range of the assay was 0.01 TCID50/well to 4.5 TCID50/well and is defined as the range of input virus that resulted in a CPE+ve proportion scoring of more than 0% but less than 100%.

Conclusion: Analysis of the four Tox revealed that inactivation was complete after incubation in 0.01% formaldehyde for 10 days at room temperature. Inactivation was achieved by days 3-4 in all lots produced, as measured by the COI assay. The COI assay is more sensitive than TCID50 potency or RNA measurements; the increased sensitivity has also been observed by LoD.

Example 5: Determining Residual Formalin Content in a Pharmaceutical Composition Materials and Methods Materials

Formaldehyde standard solution (in methanol) (982 μg/mL), DNPH, HPLC-grade acetonitrile, and phosphoric acid were purchased from Wako Pure Chemicals Co. (Tokyo, Japan). Distilled water used for diluting phosphoric acid was obtained from Otsuka Pharmaceutical (Tokushima, Japan). Alhydrogel® 2% (corresponding to 10 mg/mL aluminum) used as aluminum hydroxide gel was obtained from Brenntag (Frederikssund, Denmark). PBS was prepared in-house, and the Zika vaccine drug product containing aluminum hydroxide gel was manufactured as described below. The Zika virus was purified with various techniques after harvest. After inactivation with formaldehyde, the virus was concentrated, and the buffer was exchanged with PBS by filtration. The bulk drug substance was diluted with PBS and formulated with aluminum hydroxide gel (0.4 mg/mL aluminum) to form the final drug product.

HPLC Conditions

A Waters HPLC alliance system equipped with a UV detector (Milford, USA) and a reverse-phase column (YMC-Pack ODS-A, 4.6 mm×250 mm, 5 μm (Kyoto, Japan)) was used. A mixture of water and acetonitrile (1:1, v/v) was used as the mobile phase, the detection wavelength was set at 360 nm, and the flow rate was 1.0 mL/min. The column temperature and injection volume were 25° C. and 50 μL, respectively.

Sample Preparation

The vaccine drug product (1.2 mL) was centrifuged at 15000 rpm for 10 min, and the supernatant (1 mL) was transferred into a 2-mL HPLC glass vial purchased from Waters (Milford, USA). Next, 20 μL of 20% (v/v) phosphoric acid and 50 μL of 1.0 mg/mL DNPH solution in acetonitrile were added, and the mixture was stirred and left at room temperature for 20 min before injection.

Method Validation

According to the ICH Q2 guidelines, the method was validated in terms of specificity, linearity, accuracy, repeatability, intermediate precision, robustness, and stability of the sample. In the accuracy study, the Zika vaccine drug product and aluminum hydroxide gel solution were spiked with a specific amount of formaldehyde, and the sample was mixed well by vortex before following the procedure described in Section 2.3.

Results and Discussion Linearity and Specificity

Six standard solutions of formaldehyde (0.049, 0.098, 0.196, 0.491, 0.982, and 1.964 μg/mL) were prepared by dilution with PBS. Next, 20% (v/v) phosphoric acid and 1 mg/mL DNPH solution in acetonitrile were added to each solution, and the corresponding chromatograms are shown in FIG. 28. Clearly, the 10.4-min peak area showed linearity with the regression equation: y=1075730x+11731 (where y is the area of the 10.4-min peak and x is the concentration of formaldehyde in μg/mL) (correlation coefficient: 0.9998), indicating that it was due to HCHO-DNPH (i.e., formaldehyde derivatized with DNPH). Moreover, the peak at 5.8 min was attributed to DNPH as it was detected in all samples added with DNPH. Hence, the HCHO-DNPH peak area was used for evaluation of linearity and accuracy after subtracting the background peak area in PBS.

Accuracy and Precision (Repeatability)

The effect of aluminum hydroxide adjuvant was evaluated by recovery studies, which were carried out by spiking three samples of aluminum hydroxide (0.1, 0.4, and 1.0 mg/mL aluminum) in PBS with 0.05 μg/mL of formaldehyde in the absence of the vaccine drug substance. The average recoveries were 102% (n=3), 100% (n=3), and 100% (n=3), respectively, with low relative standard deviation (RSD) values (Table 13). The RSD of the accuracy data was calculated to evaluate the repeatability, and was found to be 1.0%, indicating that aluminum amounts up to 1.0 mg/mL did not interfere with the recovery of formaldehyde.

TABLE 13 Accuracy and repeatability evaluated using aluminum hydroxide samples spiked with 0.05 μg/mL of formaldehyde Aluminum hydroxide Average (n = 3) concentration [%] [mg/mL aluminum] (RSD [%]) 0.1 102 (0.2) 0.4 100 (0.8) 1.0 100 (0.3) Repeatability [%] (n = 9) 1.0

The accuracy of the method was evaluated by recovery studies, which were carried out by spiking the Zika vaccine drug product containing aluminum hydroxide adjuvant with three concentrations of formaldehyde (0.05, 0.10, and 1.00 μg/mL), and the average recovery results are shown in Table 14. The RSD of the accuracy data was calculated to evaluate the repeatability, and was found to be 3.7%, indicating that Zika vaccine drug products formulated with aluminum hydroxide do not interfere with the recovery of formaldehyde between 0.05 and 1.00 μg/mL.

TABLE 14 Accuracy and repeatability evaluated using Zika vaccine drug products containing aluminum hydroxide spiked with formaldehyde Spiked formaldehyde Average (n = 3) concentration [%] [mg/mL] (RSD [%]) 0.05 102 (5.6) 0.10 97 (0.3) 1.00 98 (0.7) Repeatability [%] (n = 9) 3.7

Robustness

The robustness of the method was evaluated to determine how concentration of formaldehyde in samples would be affected by variations in experimental parameters during sample preparation. Considering impact on the derivatization efficacy, concentration of DNPH and phosphoric acid were selected as the monitored parameters in this study. The effect was examined by varying the concentrations of DNPH and phosphoric acid by ±0.1 mg/mL and ±5%, respectively. Formaldehyde was determined in two development drug product lots under each condition, and the results, shown in Table 15, suggest that variations in DNPH and phosphoric acid concentrations had no significant impact on the determination of formaldehyde.

TABLE 15 Robustness of the method Concentration Concentration of Concentration of of DNPH phosphoric acid formaldehyde [μg/mL] Condition [mg/mL] [%] Lot B Lot C 1* 1.0 20 0.51 0.45 2 1.1 20 0.53 0.48 3 0.9 20 0.49 0.47 4 1.0 15 0.52 0.49 5 1.0 25 0.52 0.48 *Defined conditions of the method

Example 6: Clinical Immunogenicity and Efficacy of a Purified Inactivated Zika Virus Vaccine (PIZV) Derived from P6e Strains Sample Preparation

Four Purified Inactivated Zika Vaccine (PIZV) lots (Tox lots 1-4) were manufactured by growth in Vero cells as described above. Supernatants from 4 daily harvests (each daily harvest 1000 mL daily, totaling about 4000 mL) were purified by filtration and chromatography, concentrated and inactivated by addition of formalin to a final concentration of 0.01%. Inactivation was allowed to proceed for 10 days at 22° C., before the sample was buffer exchanged into Drug Substance Buffer (10 mM NaH2PO4, 50 mM NaCl, 6% sucrose, pH 7.4).

The inactivated Zika virus active agent is no longer able to infect host cells, which can be infected with a Zika virus, which has not been inactivated. The inactivation is determined by the following test protocol. Inactivation is acknowledged in case no plaques are detectable.

Detailed COI (Completeness of Inactivation) protocol:

-   -   1. First part of the assay: Vero (1.4E⁺⁰⁵ cells/mL) and Aedes         aegypti mosquito C6/36 (4E⁺⁰⁵ cells/mL) cells were seeded in         96-well plates two days prior to addition of the samples. The         Vero cells were cultured in DMEM+10% final FBS+2% L-glutamine+1%         penicillin/streptomycin at 37° C. C636 cells were cultured in         DMEM+10% FBS+2% L-glutamine+1% Penicillin/streptomycin+1%         nonessential amino acids at 28° C.     -   2. Three independent replicates of the 200 TCID50 control virus         (prepared in the virus back titration control test) or the DS         samples were diluted (5-fold and 10-fold dilutions) into media         containing 2% FBS.     -   3. The cells in 96-well plates were inoculated with the samples.         Prior to the infection of the cell monolayers in the 96-well         plates, the sample was vortexed to disrupt any possible         aggregation. 100 μL of each dilution was applied to each of 5         wells into two separate 96-well plates containing Vero and C636         cells, respectively.     -   4. Media alone was included in another well for each cell type         as a negative CPE control.     -   5. Plates were incubated for 6 days at the appropriate         temperature for the cell line.     -   6. Second part of the assay: To allow live virus to be further         amplified and visualized by CPE on a permissive cell line, the         entire volume of each 96-well supernatant from both Vero and         C636 cells was transferred to individual wells of 6-well plates         of Vero cells. Inoculation proceeded for 90 minutes with rocking         at 15 minutes intervals.     -   7. Medium containing 2% FBS was added to the wells and plates         were incubated for an additional 8 days for subsequent detection         of the amplified samples as a function of CPE. The inactivation         was considered to be incomplete if any of the replicates of the         DS showed CPE at the end of day 8.     -   7. The presence of live/replicating virions was visualized by         the formation of plaques or CPE on susceptible cell monolayers         after transfer to the 6-well plate, and incubation for 8 days to         allow for viral replication. The % CPE scoring in the 6-well         plates at the end of the assay was calculated as follows:         -   Each 6-well plate of Vero cells was examined for CPE by             visualization of colorimetric change, followed by             confirmation of CPE by inspection under an inverted light             microscope.         -   Each 6-well plate represented one of the replicates of the             DS dilutions prepared in the 5 and 10-fold dilutions             described above (5 wells, plus one well containing media             alone).     -    Therefore,% CPE for each replicate reflected the number of         wells with CPE out of 5 total wells per sample (120 total wells         are used per assay). Mean % CPE and standard deviation were         calculated based on three replicates of each dilution.

The amount of the purified inactivated Zika virus can be determined by a Bradford assay (Bradford et al. (1976) Anal. Biochem. 72: 248-254) using defined amounts of recombinant Zika envelope protein to establish the standard curve.

The purity of the purified Zika virus can be determined by size exclusion chromatography. In the current example, the main peak of the purified Zika virus in the size exclusion chromatography was more than 85% of the total area under the curve in the size exclusion chromatography.

The investigational vaccine (PIZV) refers to Zika purified formalin-inactivated virus formulated with 200 μg aluminum hydroxide, Al(OH)3, as adjuvant, in phosphate buffered saline solution (PBS). The final liquid formulated product is filled into single-use vials and sealed with tamper-evident seals. The investigational vaccine is administered IM (intramuscularly) as a 2-dose regimen of 0.5 mL at 2, 5, or 10 μg antigen per dose, 28 days apart.

Sodium chloride (NaCl) 0.9% solution for injection is being used as placebo. It is supplied in single-use vials. It is a sterile, clear, colorless liquid solution of sodium chloride without preservative designed for parenteral use only. The placebo is administered IM as a 2-dose regimen of 0.5 mL per dose, 28 days apart.

Test Methods

PRNT assay: Neutralizing antibody titers were determined by a plaque reduction neutralization test (PRNT) as described previously (See Protection of Rhesus monkeys against dengue virus challenge after tetravalent live attenuated dengue virus vaccination. J. Infect. Dis. 193, 1658-1665 (2006). Muthumani K, Griffin B D, Agarwal S, et al. In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine. NPJ Vaccines 2016; 1: 16021). Briefly the Zika PRNT assay was carried out according to the protocol developed by Q2 Lab Solutions Vaccines as described below.

In the Zika PRNT assay, human serum was 2-fold serially diluted from 1:5 to 1:10,240 and mixed with an equal volume of diluted Zika virus (ZIKV) (PRVABC59) to obtain a final dilution of 1:10 to 1:20,480. Neutralization was allowed to proceed 20±2 hours at 2-8° C. after which the serum/virus mixture was used to inoculate Vero E6 cells. Virus adsorption was done at 37±2° C. with humidity and CO₂ for 60±10 minutes then a methylcellulose overlay was added. The infected cells were incubated at 37±2° C. with humidity and CO₂ for 72±2 hours. Plaques were visualized by using crystal violet staining and were counted using a CTL (Cellular Technology Limited) reader. Determination of the fifty percent neutralizing titer (PRNT₅₀) was based upon the percent reduction in viral plaques in the presence of serum compared to that of the virus control without serum and was calculated by linear regression. The titers represent the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques. Acceptance was assessed by evaluating the virus control (targeting ˜60 pfu/well), cell control, positive control (PRNT₅₀ of 173-658) and negative control (PRNT₅₀<10) tested in parallel with clinical samples. Individual samples and positive control results were accepted if the correlation coefficient of the titration curve generated by linear regression is ≥0.85. Additional acceptance criteria were based on the quality of the crystal violet stain and plaques generated for the plate or run. PRNT₅₀ results are reported down to the starting dilution of the assay (1:10). PRNT₅₀ results that are above the ULOQ will be repeated at a pre-dilution to generate a result within the quantifiable range of the assay. The result from the pre-diluted sample will be multiplied by the dilution factor to generate a final result.

The Zika strain used for PRNT assay development was PRVABC59.

Immuno Outcome Measures Definitions

Sero-positivity (PRNT): titers of ≥LOD (Limit of detection)

Sero-negativity (PRNT): titers of <LOD (Limit of detection)

Seroconversion (PRNT): Post vaccination titers of ≥LOD in initially seronegative human subjects

LOD (PRNT)=10

Assigned value for below LOD=5

LLoQ (Lower Limit of Qualification, PRNT)=26

Assigned value for below LLoQ (Lower Limit of Qualification)=13

Human subjects with titers above the Upper Limit of Quantitation (ULOQ) will be retested after further dilution until a titer is obtained within the assay qualified limit of quantitation.

Reporter virus particle (RVP) neutralization assay: Neutralizing antibody titers were analyzed by titration of serum samples with a constant amount of Zika RVPs in Vero cells grown in 96-well plates. RVPs contained the prME proteins of Zika (strain SPH2012) and a Dengue-based Renilla luciferase reporter. Briefly, sera were heat inactivated at 56° C. for 30 min, diluted, and then incubated at 37° C. with RVPs. The serum/RVP mixture was then mixed with Vero cells and incubated for 72 hours at 37° C.±2° C./5% CO2 before detection with luciferase substrate. Data was analyzed using JMP11 non-linear 4 parameter analysis, normalized to a positive tracking control and effective dose 50% (EC50) was reported.

Study Description

A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, and Dose Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) in Flavivirus Naïve and Primed Healthy Adults Aged 18 to 49 Years

The study design is shown in FIG. 29. This study was designed to sequentially enroll flavivirus-naïve and flavivirus-primed healthy adults between the ages of 18 and 49 years. The two sequential cohorts are each comprised of 120 human subjects (planned) randomly allocated to one of 4 groups of 30 human subjects, to receive either one of three dosages of the PIZV vaccine or saline placebo. The vaccination regimen consists of 2 doses administered 28 days apart. The data in this example only relates to the Flavivirus naïve human subjects (n=124), further data with Flavivirus primed human subjects are to be expected. This example provides data from a first interim analysis following Day 57 (28 days post-dose 2) for the “flavivirus-naïve cohort”. Data from the flavivirus-primed cohort are not part of this interim analysis, as recruitment for this group was still ongoing at the time first interim analysis.

In summary, human subjects were randomized into four study groups, who received two doses of either placebo (saline) or purified inactivated Zika vaccine (PIZV) with a concentration of 2 μg, 5 μg and 10 μg. The study involved intramuscular injection of the vaccine (or placebo) at day 1 and day 29, with blood samples being taken on day −15, 1, 8, 29, 36, 57, 211, 393, 767 of the study. Blood samples on day −15 were used to determine Flavivirus serostatus screening and eligibility screening. Samples on day 1, 29, 57 were for immunogenicity assessment. Safety lab testing was carried out on days 8 and 36. Persistence of immunity will be assessed on day 211, 393 and 767.

Based on the data from 28 days post dose 2, the purified inactivated Zika virus vaccine (PIZV) was safe and immunogenic in Flavivirus-naïve adults aged between 18-49 yrs.

Primary Objectives

The primary objective of the study was to describe the safety of two doses of PIZV given 28 days apart and to select a dose level from three different antigen concentrations (2, 5 or 10 μg) for use in subsequent clinical studies. The primary endpoints were: the percentages of human subjects experiencing solicited local and systemic adverse events (AEs) during the 7-day period after administration of each dose of PIZV or placebo, and the percentages of human subjects experiencing non-serious unsolicited AEs and serious adverse events (SAEs) during the 28-day period after vaccination.

Secondary Objectives

The secondary objectives were to describe the immune response to the purified inactivated Zika virus vaccine (PIZV) at 28 days post dose 1 and 28 days post dose 2 in flavivirus naïve adults. The secondary endpoints related to these objectives are geometric mean titers (GMTs) of neutralizing anti-ZIKV antibodies, seropositivity rates (SPR) and seroconversion rates (SCR) at the considered timepoints.

Analysis of the data was performed by a separate set of unblinded statisticians and programmers, who had access to the individual treatment assignments. All personnel involved in the conduct of the trial were blinded to the individual human subject treatment assignments. The study team had access to the group level unblinded results only.

Study Population

A total of 124 human subjects were enrolled in the flavivirus-naïve cohort and included in the Safety Set (SS), comprised of all randomized human subjects who have received at least one dose of PIZV or placebo. Among those, 118 (95.2% of the SS) were included in the Full Analysis Set (FAS) of randomized human subjects who had received at least one dose of the investigational vaccine (PIZV)/placebo, provided valid serology results at baseline and at least once post-vaccination. One hundred and thirteen (113) human subjects (91.1% of the SS) were included in the Per Protocol Set (PPS) of human subjects in the FAS who had no major protocol violations relevant for the immunogenicity analysis. The analysis sets are presented in Table 16.

TABLE 16 Analysis sets Number of Human subjects (%) Placebo 2 μg PIZV 5 μg PIZV 10 μg PIZV Total (N = 30) (N = 31) (N = 31) N = 32) (N = 124) Safety Set (SS) 30 (100%) 31 (100%) 31 (100%) 32 (100%) 124 (100%) Full Analysis 29 (96.7%) 28 (90.3%) 31 (100%) 30 (93.8%) 118 (95.2%) Set (FAS) Per-Protocol 28 (93.3%) 26 (83.9%) 29 (93.5%) 30 (93.8%) 113 (91.1%) Set (PPS) Safety Set = all randomized human subjects who received at least one (1) dose of PIZV or placebo Full Analysis Set = all randomized human subjects who received at least one dose of PIZV/placebo and provided valid baseline and at least one post-vaccination serology result Per Protocol Set = all human subjects in the FAS who had no major protocol violations

Human subjects in the SS were 35.3±8.91 years of age (mean±standard deviation), and were distributed as 28.2% in the 18-29 years age-range and 71.8% in the 30-49 years age-range. Women represented 54.8% of the cohort. Study participants were White (81.5%), Black (14.5%), and “Non-Hispanic” (93.5%) regarding race and ethnicity. The mean BMI in the SS was 27.5±4.05 (mean±standard deviation). Demographic characteristics (age, sex, height, weight, BMI and ethnicity) were overall similar across the four study groups. Women were more represented in the placebo group, where they constituted 66% of the study participants, than in the other groups, where gender distribution was more balanced. The demographics and baseline characteristics are presented in Table 17.

Safety laboratory parameters and vital signs were checked at study entry as part of inclusion criteria. These specified that vital signs had to be within normal limits (i.e., below Grade 1 as indicated in the FDA Toxicity Grading Scale) and that safety laboratory tests had to be within normal limits or not be above Grade 1 as defined in the FDA Toxicity Grading Scale.

TABLE 17 Demographic and Baseline Characteristics (Safety Set) Number of Human subjects (%) Placebo 2 ug 5 ug 10 ug Total (N = 30) (N = 31) (N = 31) (N = 32) (N = 124) Age (Years) n 30 31 31 32 124 Mean (SD) 36.5 (9.00) 34.9 (9.52) 35.8 (8.86) 34.1 (8.50) 35.3 (8.91) Median 39.5 36.0 36.0 33.5 36.0 Minimum, 18, 49 18, 48 20, 49 20, 49 18, 49 Maximum Age (years) (n [%]) 18-29   8 (26.7)   9 (29.0)   9 (29.0)   9 (28.1)   35 (28.2) 30-49   22 (73.3)   22 (71.0)   22 (71.0)   23 (71.9)   89 (71.8) Sex (n [%]) Male   10 (33.3)   15 (48.4)   13 (41.9)   18 (56.3)   56 (45.2) Female   20 (66.7)   16 (51.6)   18 (58.1)   14 (43.8)   68 (54.8) Ethnicity (n [%]) Hispanic   1 (3.3)   1 (3.2)   2 (6.5)   4 (12.5)   8 (6.5) or Latino Not-Hispanic   29 (96.7)   30 (96.8)   29 (93.5)   28 (87.5)  116 (93.5) or Latino Not Reported  0  0  0  0  0 Unknown  0  0  0  0  0 Race (n [%]) American   2 (6.7)  0  0  0   2 (1.6) Indian or Alaskan Native Asian  0  0  0  0  0 Black or   6 (20.0)   5 (16.1)   3 (9.7)   4 (12.5)   18 (14.5) African American Native  0  0  0  0  0 Hawaiian or Other Pacific Islander White   22 (73.3)   26 (83.9)   26 (83.9)   27 (84.4)  101 (81.5) Multiracial  0  0   2 (6.5)   1 (3.1)   3 (2.4) BMI (kg/m²) n 30 31 31 32 124 Mean (SD) 28.169 27.527 27.541 26.750 27.485 (4.0388) (4.7632) (3.7165) (3.6511) (4.0452) Median 28.861 27.900 27.831 25.965 27.607 Minimum, 20.86, 20.13, 18.84, 18.80, 18.80, Maximum 34.64 34.83 34.14 34.37 34.83 BMI = Weight (kg)/height² (m²). Note 1: Age is calculated using the Date of Informed Consent. Note 2: Human subject included in Multiracial Category only if multiple Race categories selected.

Safety/Reactogenicity

The overall reporting incidence of solicited local adverse events (AEs) was higher in the groups that received the vaccine (PIZV) than in the placebo group. Pain was the most frequently reported solicited AE at the injection site. After dose 1, pain was experienced by 30.0% to 38.7% of human subjects in the PIZV groups compared to 13.8% in the placebo group. After dose 2, incidences of pain were similar to those following dose 1: 29.6% to 40% in the PIZV groups, and 14.3% in the placebo group. Intensity of pain was reported as mild after dose 1 and mild to moderate after dose 2, with 2 human subjects in the 5 μg PIZV group (6.7%) and one human subject in the 10 μg PIZV group (3.3%) reporting moderate pain. Other solicited local AEs (erythema, swelling and induration) were reported by not more than 9.7% of the human subjects.

The onset of pain occurred on day 1 for 90% of the human subjects or day 2 (for 3 human subjects). Pain was not reported beyond day 5 by any human subject in the placebo or PIZV groups.

Solicited systemic AEs of any nature were reported by 30% to 48.4% of the human subjects across the PIZV groups and by 41.4% in the Placebo group after dose 1. After dose 2, incidences were 10% to 33.3% across the PIZV groups and 27.6% in the Placebo group. Overall 81.3% (dose 1) and 75% (dose 2) of the solicited systemic AEs were judged as related to study vaccination. After both doses, the most reported systemic events were headache, fatigue and myalgia.

Most systemic AEs were reported as mild, i.e. not interfering with daily activity. A few occurrences were moderate in intensity:

after dose 1, for 6.7-12.9% of human subjects in the PIZV groups and 17.2% of placebo recipients;

after dose 2, for 0-3.3% of human subjects across the PIZV groups and 10.3% in the placebo group.

There was a single report of a severe AE: one human subject in the placebo group experienced fever. This study participant presented a temperature of 39.4° C., measured orally, 4 days after receiving the second study vaccination. This fever was not judged as study-related by the investigator.

Solicited systemic AEs were variably reported throughout the 7-day period in the four groups. The onset of events for fever, fatigue, arthralgia and myalgia was mainly during the 2 days following vaccination and was variable for headache and malaise. Fever was reported during the 2 days following vaccination, except for the human subject reporting severe fever in the placebo group on day 4.

The incidence of solicited local and systemic adverse events 7 days after vaccination are shown in Table 18.

TABLE 18 Incidence of solicited local and systemic adverse events 7 days after vaccination (Safety set) Dose 1 Dose 2 2 μg 5 μg 10 μg 2 μg 5 μg 10 μg Placebo PIZV PIZV PIZV Placebo PIZV PIZV PIZV (N = 30) (N = 31) (N = 31) (N = 32) (N = 30) (N = 31) (N = 31) (N = 32) Local AEs n (%) Any 4 (13.8) 9 (30.0) 12 (38.7) 13 (41.9)  5 (17.9) 8 (29.6) 11 (36.7) 12 (40.0) Pain 4 (13.8) 9 (30.0) 10 (32.3) 12 (38.7)  4 (14.3) 8 (29.6) 11 (36.7) 12 (40.0) Erythema 0 0 0 1 (3.2) 1 (3.6) 0 1 (3.3) 1 (3.3) Swelling 0 0 0 0 0 0 2 (6.7) 0 Induration 0 0 3 (9.7) 2 (6.5) 0 0 1 (3.3) 0 Systemic AEs n (%) Any 12 (41.4)  9 (30.0) 12 (38.7) 15 (48.4)  8 (27.6) 9 (33.3)  3 (10.0)  8 (26.7) Fever 1 (3.4)  0 0 1 (3.2) 2 (7.1) 0 0 0 Headache 9 (31.0) 5 (16.7)  8 (25.8)  4 (12.9)  3 (10.3) 4 (14.8) 1 (3.3)  6 (20.0) Fatigue 6 (20.7) 7 (23.3)  6 (19.4) 10 (32.3)  6 (20.7) 3 (11.1) 2 (6.7)  5 (16.7) Arthralgia 1 (3.4)  1 (3.3)  1 (3.2) 3 (9.7) 1 (3.4) 2 (7.4)  0 1 (3.3) Myalgia 3 (10.3) 3 (10.0)  5 (16.1)  4 (12.9) 2 (6.9) 2 (7.4)  1 (3.3) 2 (6.7) Malaise 4 (13.8) 2 (6.7)  2 (6.5)  4 (12.9) 2 (6.9) 0 0  3 (10.0) N = number of human subjects with information available; n (%) = number (percentage) of human subjects reporting a specific AE.

In total 30.6% of the human subjects reported unsolicited AEs (not including prolonged solicited AEs) in the 28 days following any dose: 21.9-38.7% in the PIZV groups and 36.7% in the placebo group. These AEs were mainly infections, infestations (13.7%) and nervous system disorders (3.2%: headache, migraine, dizziness). All were mild to moderate in intensity.

Unsolicited AEs were considered as related to the study vaccination for three human subjects (2.4%). The events reported were:

at post dose 1, dizziness for one human subject in the 5 μg PIZV group and flushing for one human subject in the 2 μg PIZV group;

at post dose 2, eye pruritus and lacrimation increased for one human subject in the 10 μg PIZV group.

These were mild to moderate in intensity, started on day 1 or 2 after vaccination, had a duration of 1 to 3 days and were all resolved.

One human subject discontinued with the study vaccination due to a headache after dose 1. This human subject received PIZV and experienced the headache 1 day after vaccination. The headache was resolved 36 days after its onset. No serious adverse event (SAE) was reported during the period from dose 1 up to 28 days post dose 2.

The few changes from the baseline observed for blood safety laboratory parameters in the 7 days following vaccination, e.g. from normal to mild or from mild to moderate AEs, occurred in comparable percentages of human subjects across the four groups. Urinalysis parameters were either normal at all time-points or the grading category was similar across groups and visits.

Immunogenicity

Table 19 presents the geometric antibody titers of Zika virus neutralizing antibodies (EC50) as measured by PRNT as well as seropositivity rates and seroconversion rates after each vaccine dose.

The PIZV vaccine was immunogenic in flavivirus-naive adults. All human subjects were seronegative at baseline. Vaccination of human subjects initially seronegative for antibodies against Zika virus elicited seropositivity in all human subjects after two doses of PIZV vaccine of any dosage: seroconversion rates ranged from 69.23% to 96.43% post-dose 1 and were 100% post-dose 2. All human subjects in the placebo group remained seronegative throughout the period considered.

A dose-ranging effect was observed on seropositivity rates post-dose 1 and on GMTs after each dose. After the first dose, almost all human subjects (96.4%) who had received the 10 μg PIZV dose had mounted neutralizing antibodies against the Zika virus. The second dose led to a more than 10-fold increase in GMT from dose 1, in the three PIZV groups.

TABLE 19 Seropositivity, seroconversion rates and GMTs (Geometric mean titers) of Zika virus neutralizing antibodies (EC₅₀) (PRNT) before and 28 days after administration of each dose of PIZV (Per Protocol Set) Placebo 2 μg PIZV 5 μg PIZV 10 μg PIZV (N = 28) (N = 26) (N = 29) (N = 30) Sero- Pre-dose 0 0 0 0 positivity 1 rate Post-dose 0 69.23 (48.21, 82.14 (63.11, 96.43 (81.65, (95% CI) 1 85.67) 93.94) 99.91) Post-dose 0 100 (85.75, 100 (87.66, 100 (87.66, 2 100.00) 100.00) 100.00) Sero- Post-dose 0 69.23 (48.21, 82.14 (63.11, 96.43 (81.65, conversion 1 85.67) 93.94) 99.91) rate Post-dose 0 100 (85.75, 100 (87.66, 100 (87.66, (95% CI) 2 100.00) 100.00) 100.00) GMTs Pre-dose 5.00 5.00 5.00 5.00 (95% CI) 1 Post-dose 5.00 38.06 (17.53, 93.76 (44.34, 291.41 (161.74, 1 82.66) 198.30) 525.06) Post-dose 5.00 1100.75 (741.07, 1992.33 (1401.28, 3689.89 (2676.75, 2 1635.00) 2832.70) 5086.49) N = number of human subjects in the PPS with PRNT data available; Seropositivity is defined as titer ≥10; Seroconversion is defined as: seronegative human subjects at baseline (titer <10) have titer ≥10 post-vaccination; Results <10 are assigned a titer of 5; Titers ≥10 (limit of detection) and <26 (lower limit of quantification) are assigned a value of 13.

The Geometric mean titers determined using PRNT, according to table 19, are shown graphically in FIG. 30. The percentage of human subjects achieving seroconversion determine using PRNT according to table 19, are shown graphically in FIG. 31.

The distribution of neutralization titers, after dose 1 and after dose 2, are shown in reverse cumulative distribution curves in FIGS. 32 and 33 respectively.

In addition to measuring immune response with the PRNT assay, the samples were also tested with the RVP neutralization assay. Table 20 presents the geometric antibody titers of Zika virus neutralizing antibodies (EC50) as measured by the RVP assay. The RVP assay results show a similar dose-ranging effect of the PRNT data, with gradually higher GMTs with increasing PIZV doses.

TABLE 20 GMTs (Geometric mean titers) of Zika virus neutralizing antibodies (EC₅₀) (RVP) before and 28 days after vaccination (Per Protocol Set) Placebo 2 μg PIZV 5 μg PIZV 10 μg PIZV Group (N = 27) (N = 26) (N = 28) (N = 30) GMTs Pre-dose 1 34 (27, 43)  34 (26, 44)  32 (27, 39)   46 (39, 53) (95% CI) Post-dose 1 28 (22, 36)  360 (242, 536)  656 (442, 972)  1310 (875, 1961) Post-dose 2 31 (25, 40) 3148 (1988, 4986) 6212 (4126, 9354) 13604 (9560, 19359) N = number of human subjects in the PPS with RVP data available.

Conclusion

The PIZV vaccine was well tolerated and safe for all antigen doses evaluated in the flavivirus-naive cohort. Solicited systemic AEs were reported in all groups with no apparent increase with increasing dose strength and intensity was mild to moderate. Local solicited AEs reported were also mild to moderate in intensity across the groups. Unsolicited symptoms were reported with similar frequencies in the four study groups. Overall, the vaccine was immunogenic in flavivirus-naive human subjects and a positive dose-ranging response was observed.

Further Items of the Invention:

-   -   1. Vaccine or immunogenic composition for use in a method of         treating or preventing, in particular preventing Zika virus         infection in a human subject population in need thereof,         comprising administering to individual human subjects of said         human subject population the vaccine or immunogenic composition         comprising antigen from a Zika virus, wherein the vaccine or         immunogenic composition is administered as a single dose or         prime administration, and wherein the administration of the         vaccine or immunogenic composition induces 14 and/or 28 days         after the single dose or prime administration geometric mean         neutralizing antibody titers in a population of at least 20         flavivirus naïve human subjects and/or in a population of at         least 20 Zika virus seronegative human subjects of greater than         10, or greater than 50, or greater than 100, or greater than         200, or greater than 250, as determined by the plaque reduction         neutralization test (PRNT).     -   2. Vaccine or immunogenic composition for use in a method of         treating or preventing, in particular preventing Zika virus         infection in a human subject population in need thereof,         comprising administering to individual human subjects of said         human subject population the vaccine or immunogenic composition         comprising antigen from a Zika virus, wherein the vaccine or         immunogenic composition is administered as single dose or prime         administration and wherein the administration of the vaccine or         immunogenic composition induces 14 and/or 28 days after the         single dose or prime administration a seroconversion rate of at         least 25%, at least 30%, at least 40%, at least 50%, at least         60%, at least 70%, at least 75%, at least 80%, at least 85%, at         least 86%, at least 87%, at least 88%, at least 89%, or at least         90% in a population of at least 20 Zika virus seronegative human         subjects, as determined by the plaque reduction neutralization         test (PRNT).     -   3. Vaccine or immunogenic composition for use in a method of         treating or preventing, in particular preventing Zika virus         infection in a human subject population in need thereof,         comprising administering to individual human subjects of said         human subject population the vaccine or immunogenic composition         comprising antigen from a Zika virus, wherein the vaccine or         immunogenic composition is administered as single dose or prime         administration and wherein the administration of the vaccine or         immunogenic composition induces 14 and/or 28 days after the         single dose or prime administration a seropositivity rate of at         least 25%, at least 30%, at least 40%, at least 50%, at least         60%, at least 70%, at least 75%, at least 80%, at least 85%, at         least 86%, at least 87%, at least 88%, at least 89%, or at least         90% in a population of at least 20 Zika virus seronegative human         subjects or in a population of at least 20 Flavivirus naïve         human subjects, as determined by the plaque reduction         neutralization test (PRNT).     -   4. Vaccine or immunogenic composition for use in a method of         treating or preventing, in particular preventing Zika virus         infection in a human subject population in need thereof,         comprising administering to individual human subjects of said         human subject population the vaccine or immunogenic composition         comprising antigen from a Zika virus, wherein the vaccine or         immunogenic composition is administered as a single dose         administration or multi dose administration including at least a         first (prime) and a second (boost) administration and wherein         the administration of the vaccine or immunogenic composition         until 7 days after the administration induces systemic side         effects in less than 50% of a human subject population of at         least 20 flavivirus naïve human subjects or in a population of         at least 20 Zika virus seronegative human subjects.     -   5. Vaccine or immunogenic composition for use in a method of         treating or preventing, in particular preventing Zika virus         infection in a human subject population in need thereof,         comprising administering to individual human subjects of said         human subject population the vaccine or immunogenic composition         comprising antigen from a Zika virus obtainable by a method for         inactivating a Zika virus preparation comprising:         -   (a) isolating the Zika virus preparation from one or more             cells cultured in vitro, wherein the cells are used to             produce the Zika virus preparation, wherein isolating the             Zika virus preparation comprises one or more steps selected             from: (i) depth filtration, (ii) buffer exchange and/or             dilution; (iii) ion exchange chromatography; and         -   (b) treating the Zika virus preparation with formaldehyde,             optionally with formaldehyde, wherein the numerical result             of the multiplication of the formaldehyde concentration as             measured in % (w/v) with the period of incubation with             formaldehyde as measured in days is 0.025 to 0.5.     -   6. The vaccine or immunogenic composition of any one of items 1         to 5, wherein the vaccine or immunogenic composition is         administered as a first (prime) and a second (boost)         administration about 1 to about 16 weeks apart, and wherein the         administration of the vaccine or immunogenic composition induces         14 and/or 28 days after the boost administration geometric mean         neutralizing antibody titers in a population of at least 20         flavivirus naïve human subjects and/or in a population of at         least 20 Zika virus seronegative human subjects of greater than         300, or greater than 500, or greater than 1000, or greater than         1500, or greater than 2000, or greater than 3000, as determined         by the plaque reduction neutralization test (PRNT).     -   7. The vaccine or immunogenic composition of any one of items 1         to 6, wherein the vaccine or immunogenic composition is         administered as a first (prime) and a second (boost)         administration about 1 to about 16 weeks apart and wherein the         administration of the vaccine or immunogenic composition induces         14 and/or 28 days after the boost administration         -   a seroconversion rate of at least 70%, at least 75%, at             least 80%, at least 85%, at least 86%, at least 87%, at             least 88%, at least 89%, or at least 90%, at least 91%, at             least 92%, at least 93%, at least 94%, at least 95%, at             least 96%, at least 97%, at least 98%, at least 99% or 100%             in a population of at least 20 Zika virus seronegative human             subjects, as determined by the plaque reduction             neutralization test (PRNT),         -   and/or         -   a seropositivity rate of at least 70%, at least 75%, at             least 80%, at least 85%, at least 86%, at least 87%, at             least 88%, at least 89%, or at least 90%, at least 91%, at             least 92%, at least 93%, at least 94%, at least 95%, at             least 96%, at least 97%, at least 98%, at least 99% or 100%             in a population of at least 20 Zika virus seronegative human             subjects or in a population of at least 20 Flavivirus naïve             human subjects, as determined by the plaque reduction             neutralization test (PRNT).     -   8. The vaccine or immunogenic composition of any one of items 1         to 7, wherein the administration of the vaccine or immunogenic         composition until 7 days after the administration induces         headache symptoms in less than 29% of a human subject population         of at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects.     -   9. The vaccine or immunogenic composition of any one of items 1         to 8, wherein the administration of the vaccine or immunogenic         composition until 7 days after the administration induces fever         in 4% or less, and/or fatigue in 33% or less, and/or arthralgia         in 10% or less, and/or myalgia in 17% or less, and/or malaise in         15% or less of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   10. The vaccine or immunogenic composition of any one of items 1         to 9, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces systemic side effects in less than 40% of         a human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   11. The vaccine or immunogenic composition of any one of items 1         to 10, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces         -   at least 40% less, or at least 45% less fatigue, and/or         -   no more fever, and/or         -   no more, or at least 10% less, or at least 20% less, or at             least 25% less myalgia, and/or         -   no more, or at least 10% less, or at least 20% less malaise     -    compared to 7 days after the prime administration in a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   12. The vaccine or immunogenic composition of any one of items 1         to 11, wherein the vaccine or immunogenic composition is         administered as a multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces fever in less than 4% or in 0% of a human         subject population of at least 20 flavivirus naïve human         subjects or at least 20 Zika virus seronegative human subjects.     -   13. The vaccine or immunogenic composition of any one of items 1         to 12, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces fatigue in less than 19% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   14. The vaccine or immunogenic composition of any one of items 1         to 13, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces myalgia in less than 12% or less than 8%         of a human subject population of at least 20 flavivirus naïve         human subjects or of at least 20 Zika virus seronegative human         subjects.     -   15. The vaccine or immunogenic composition of any one of items 1         to 14, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces malaise in less than 13% or in 10% or         less of a human subject population of at least 20 flavivirus         naïve human subjects or of at least 20 Zika virus seronegative         human subjects.     -   16. The vaccine or immunogenic composition of any one of items 1         to 15, wherein the vaccine or immunogenic composition is         administered as a multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces headache symptoms in 20% or less, and         arthralgia in 8% or less, and fever in less than 4%, and fatigue         in less than 19%, and myalgia in less than 12%, and malaise in         less than 13% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   17. The vaccine or immunogenic composition of any one of claims         1 to 16, wherein the vaccine or immunogenic composition         comprises a dose of 1 μg to 40 μg of the antigen, wherein the         antigen is an inactivated whole virus.     -   18. The vaccine or immunogenic composition of claim 17, the Zika         virus having a mutation at position 98 of SEQ ID NO: 1, or at a         position corresponding to position 98 of SEQ ID NO: 1.     -   19. The vaccine or immunogenic composition of any one of claims         1 to 18, wherein the antigen is purified, and wherein the main         peak of the purified antigen in the size exclusion         chromatography is more than 85% of the total area under the         curve in the size exclusion chromatography.     -   20. The vaccine or immunogenic composition of any one of claims         1 to 19, wherein the vaccine or immunogenic composition is         administered to human subjects from a Zika endemic region,         optionally subject to an outbreak.     -   21. The vaccine or immunogenic composition of any one of claims         1 to 20, wherein the vaccine or immunogenic composition is         administered to human subjects from a Zika non-endemic region         travelling to an endemic region.     -   22. The vaccine or immunogenic composition of any one of claims         1 to 21, wherein the vaccine or immunogenic composition is         administered to human subjects 18 to 29 years of age, in         particular women of childbearing potential.     -   23. The vaccine or immunogenic composition of any one of claims         1 to 21, wherein the vaccine or immunogenic composition is         administered to human subjects 30 to 49 years of age, in         particular women of childbearing potential.     -   24. The vaccine or immunogenic composition of any one of items 1         to 23, wherein the vaccine or immunogenic composition is         administered as a single dose administration or multi dose         administration including at least a first (prime) and a second         (boost) administration wherein the vaccine or immunogenic         composition comprises a dose of about 5 μg of purified         inactivated whole virus.     -   25. The vaccine or immunogenic composition of claim 24 wherein         the administration of the vaccine or immunogenic composition         until 7 days after the single dose or prime administration         induces systemic side effects in less than 50%, or in less than         45%, or in less than 40% of a human subject population of at         least 20 flavivirus naïve human subjects or of at least 20 Zika         virus seronegative human subjects and/or wherein the         administration of the vaccine or immunogenic composition until 7         days after the boost administration induces systemic side         effects in less than 40%, or in less than 35%, or in less than         30%, or in less than 25%, or in less than 20%, or in less than         15% of a human subject population of at least 20 flavivirus         naïve human subjects or of at least 20 Zika virus seronegative         human subjects.     -   26. The vaccine or immunogenic composition of claim 24 or 25,         wherein the administration of the vaccine or immunogenic         composition until 7 days after the single dose or prime         administration induces fever in less than 3%, or 0%, of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces fever in less than 4%, or in less than         3%, or 0% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   27. The vaccine or immunogenic composition of any one of items         24 to 26, herein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces headache in less than 29% of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces headache in less than 20%, or in less         than 15%, or in less than 10%, or in less than 5% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   28. The vaccine or immunogenic composition of any one of items         24 to 27, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces fatigue in less than 30%, or in         less than 25%, or in less than 20% of a human subject population         of at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects and/or wherein the         administration of the vaccine or immunogenic composition until 7         days after the boost administration induces fatigue in less than         20%, or in less than 15%, or in less than 10% of a human subject         population of at least 20 flavivirus naïve human subjects or of         at least 20 Zika virus seronegative human subjects.     -   29. The vaccine or immunogenic composition of any one of items         24 to 28, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces arthralgia in less than 4% of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces arthralgia in less than 5%, or in less         than 2%, or in 0% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   30. The vaccine or immunogenic composition of any one of items         24 to 29, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces myalgia in 17% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces myalgia in less than 12%, or in less than         10%, or in less than 5% of a human subject population of at         least 20 flavivirus naïve human subjects or of at least 20 Zika         virus seronegative human subjects.     -   31. The vaccine or immunogenic composition of any one of items         24 to 30, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces malaise in less than 10% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces malaise in less than 14%, or in less than         10%, or in less than 5%, or 0% of a human subject population of         at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects.     -   32. The vaccine or immunogenic composition of any one of items         24 to 31, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces         -   at least 70% less, or at least 60% less, or at least 50%             less, or at least 40% less, or at least 35% less, or at             least 30% less systemic side effects, and/or         -   no increase in fever, and/or         -   at least 80% less, or at least 70% less, or at least 60%             less, or at least 50% less, or at least 45% less headache,             and/or         -   at least 60% less, or at least 55% less, or at least 50%             less, or at least 45% less, or at least 40% less fatigue,             and/or         -   no increase in arthralgia, or at least 80% less, or at least             60% less, or at least 40% less, or at least 20% less, or at             least 10% less arthralgia, and/or         -   no increase in myalgia, or at least 70% less, or at least             60% less, or at least 40% less, or at least 20% less, or at             least 10% less myalgia, and/or         -   no increase in malaise, or at least 80% less, or at least             60% less, or at least 40% less, or at least 20% less, or at             least 10% less malaise         -   compared to 7 days after the prime administration in a human             subject population of at least 20 flavivirus naïve human             subjects or of at least 20 Zika virus seronegative human             subjects.     -   33. The vaccine or immunogenic composition of any one of items 1         to 23, wherein the vaccine or immunogenic composition is         administered as a single dose administration or multi dose         administration including at least a first (prime) and a second         (boost) administration wherein the vaccine or immunogenic         composition comprises a dose of about 10 μg of purified         inactivated whole virus.     -   34. The vaccine or immunogenic composition of claim 33, wherein         the administration of the vaccine or immunogenic composition         until 7 days after the single dose or prime administration         induces systemic side effects in less than 50% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces systemic side effects in less than 40%,         or in less than 35%, or in less than 30% of a human subject         population of at least 20 flavivirus naïve human subjects or of         at least 20 Zika virus seronegative human subjects.     -   35. The vaccine or immunogenic composition of claim 33 or 34,         wherein the administration of the vaccine or immunogenic         composition until 7 days after the single dose or prime         administration induces fever in less than 4% of a human subject         population of at least 20 flavivirus naïve human subjects or of         at least 20 Zika virus seronegative human subjects and/or         wherein the administration of the vaccine or immunogenic         composition until 7 days after the boost administration induces         fever in less than 4%, or in less than 3%, or 0% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   36. The vaccine or immunogenic composition of any one of items         33 to 35, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces headache in less than 29%, or in         less than 25%, or in less than 20%, or in less than 15% of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces headache in 20% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   37. The vaccine or immunogenic composition of any one of items         33 to 36, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces fatigue in 33% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces fatigue in less than 20% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   38. The vaccine or immunogenic composition of any one of items         33 to 37, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces arthralgia in 10% or less of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces arthralgia in less than 5% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   39. The vaccine or immunogenic composition of any one of items         33 to 38, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces myalgia in 17% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces myalgia in less than 12%, or in less than         10% of a human subject population of at least 20 flavivirus         naïve human subjects or of at least 20 Zika virus seronegative         human subjects.     -   40. The vaccine or immunogenic composition of any one of items         33 to 39, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces malaise in 15% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces malaise in less than 13%, or in 10% or         less of a human subject population of at least 20 flavivirus         naïve human subjects or of at least 20 Zika virus seronegative         human subjects.     -   41. The vaccine or immunogenic composition of any one of items         33 to 40, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces         -   at least 40% less, or at least 35% less, or at least 30%             less, or at least 25% less systemic side effects, and/or         -   no increase in fever, or at least 80% less, or at least 60%             less, or at least 40% less, or at least 20% less, or at             least 10% less fever, and/or         -   at least 45% less, or at least 40% less fatigue, and/or         -   no increase in arthralgia, or at least 65% less, or at least             60% less, or at least 40% less, or at least 20% less, or at             least 10% less arthralgia, and/or         -   no increase in myalgia, or at least 45% less, or at least             40% less, or at least 20% less, or at least 10% less             myalgia, and/or         -   no increase in malaise, or at least 20% less, or at least             10% less malaise         -   compared to 7 days after the prime administration in a human             subject population of at least 20 flavivirus naïve human             subjects or of at least 20 Zika virus seronegative human             subjects.     -   42. The vaccine or immunogenic composition of any one of items 1         to 23, wherein the vaccine or immunogenic composition is         administered as a single dose administration or multi dose         administration including at least a first (prime) and a second         (boost) administration wherein the vaccine or immunogenic         composition comprises a dose of about 2 μg of purified         inactivated whole virus.     -   43. The vaccine or immunogenic composition of claim 42, wherein         the administration of the vaccine or immunogenic composition         until 7 days after the single dose or prime administration         induces systemic side effects in less than 50%, or in less than         45%, or in less than 40%, or in less than 35% of a human subject         population of at least 20 flavivirus naïve human subjects or of         at least 20 Zika virus seronegative human subjects and/or         wherein the administration of the vaccine or immunogenic         composition until 7 days after the boost administration induces         systemic side effects in less than 40%, or in less than 35% of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   44. The vaccine or immunogenic composition of claim 42 or 43,         wherein the administration of the vaccine or immunogenic         composition until 7 days after the single dose or prime         administration induces fever in less than 3%, or 0%, of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces fever in less than 4%, or in less than         3%, or 0% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   45. The vaccine or immunogenic composition of any one of items         42 to 44, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces headache in less than 29%, or in         less than 25%, or in less than 20% of a human subject population         of at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects and/or wherein the         administration of the vaccine or immunogenic composition until 7         days after the boost administration induces headache in less         than 20%, or in less than 15% of a human subject population of         at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects.     -   46. The vaccine or immunogenic composition of any one of items         42 to 45, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces fatigue in less than 30%, or in         less than 25% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects and/or wherein the administration of         the vaccine or immunogenic composition until 7 days after the         boost administration induces fatigue in less than 20%, or in         less than 15% of a human subject population of at least 20         flavivirus naïve human subjects or of at least 20 Zika virus         seronegative human subjects.     -   47. The vaccine or immunogenic composition of any one of items         42 to 46, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces arthralgia in less than 4% of a         human subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces arthralgia in less than 8% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects.     -   48. The vaccine or immunogenic composition of any one of items         42 to 47, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces myalgia in 17% or less of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces myalgia in less than 12%, or in less than         10% of a human subject population of at least 20 flavivirus         naïve human subjects or of at least 20 Zika virus seronegative         human subjects.     -   49. The vaccine or immunogenic composition of any one of items         42 to 48, wherein the administration of the vaccine or         immunogenic composition until 7 days after the single dose or         prime administration induces malaise in less than 10% of a human         subject population of at least 20 flavivirus naïve human         subjects or of at least 20 Zika virus seronegative human         subjects and/or wherein the administration of the vaccine or         immunogenic composition until 7 days after the boost         administration induces malaise in less than 13%, or in less than         10%, or in less than 5%, or 0% of a human subject population of         at least 20 flavivirus naïve human subjects or of at least 20         Zika virus seronegative human subjects.     -   50. The vaccine or immunogenic composition of any one of items         42 to 49, wherein the vaccine or immunogenic composition is         administered as multiple doses in a first (prime) and a second         (boost) administration and wherein the administration of the         vaccine or immunogenic composition until 7 days after the boost         administration induces         -   no increase in fever, and/or         -   at least 50% less, or at least 45% less, or at least 40%             less fatigue, and/or         -   no increase in myalgia, or at least 20% less, or at least             10% less myalgia, and/or         -   no increase in malaise, or at least 80% less, or at least             60% less, or at least 40% less, or at least 20% less, or at             least 10% less malaise         -   compared to 7 days after the prime administration in a human             subject population of at least 20 flavivirus naïve human             subjects or of at least 20 Zika virus seronegative human             subjects.     -   51. The vaccine or immunogenic composition of any one of items 5         to 50, wherein the cells are non-human cells.     -   52. The vaccine or immunogenic composition of claim 51, wherein         the cells are Vero cells.     -   53. The vaccine or immunogenic composition of any one of items 5         to 52, wherein the Zika virus preparation is treated with         formaldehyde at a concentration of 0.005% (w/v) to 0.02% (w/v).     -   54. The vaccine or immunogenic composition of any one of items 5         to 53, wherein the Zika virus preparation is treated for eight         to twelve days.     -   55. The vaccine or immunogenic composition of claim 54, wherein         the Zika virus preparation is treated for ten days.     -   56. The vaccine or immunogenic composition of any one of items 5         to 55, wherein the Zika virus preparation is treated at a         temperature of 15° C. to 30° C.     -   57. The vaccine or immunogenic composition of claim 56, wherein         the Zika virus preparation is treated at a temperature of 22° C.     -   58. The vaccine or immunogenic composition of any one of items 5         to 57, further comprising a step (c) of determining the         completeness of inactivation.     -   59. The vaccine or immunogenic composition of claim 58, wherein         step (c) comprises:         -   (i) inoculating cultured insect cells with a Zika virus             preparation treated according to step (b) and incubating the             insect cells for a first period of time, thereby producing             an insect cell supernatant;         -   (ii) inoculating cultured mammalian cells with the insect             cell supernatant produced in (i) and incubating the             mammalian cells for a second period of time; and         -   (iii) determining whether the Zika virus preparation             contains a residual replicating virus that produces a             cytopathic effect on the mammalian cells.     -   60. The vaccine or immunogenic composition of claim 59, wherein         the insect cells are selected from CCL-125 cells, Aag-2 cells,         RML-12 cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t. GRIP-1         cells, A.t. GRIP-2 cells, A.t. GRIP-3 cells, UM-AVE1 cells,         Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43         cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells, such         as C6/36 cells.     -   61. The vaccine or immunogenic composition of claim 59 or 60,         wherein the first period of time is 3 to 7 days.     -   62. The vaccine or immunogenic composition of any one of items         59 to 61, wherein the mammalian cells are selected from VERO         cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK         (NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219 as         described in WO97/37001) cells, BHK21-F cells, HKCC cells, and         Chinese hamster ovary cells (CHO cells), such as VERO cells.     -   63. The vaccine or immunogenic composition of any one of items         59 to 62, wherein the second period of time is 3 to 14 days.     -   64. The vaccine or immunogenic composition of any one of items 5         to 63, further comprising a step (d) of neutralizing the         formaldehyde-treated Zika virus preparation with sodium         metabisulfite.     -   65. The vaccine or immunogenic composition of claim 64, wherein         the formaldehyde-treated Zika virus preparation is neutralized         at least five, at least seven, at least nine, at least 11, or at         least 14 days after formaldehyde treatment.     -   66. The vaccine or immunogenic composition of any one of items 1         to 65, wherein the vaccine or immunogenic composition has a         residual formaldehyde content of less than 50 μg/ml. 

1-68. (canceled)
 69. A method of treating or preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as a single dose or prime administration, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or in a population of at least 20 Zika virus seronegative human subjects of greater than 10, as determined by the plaque reduction neutralization test (PRNT).
 70. A Method of treating or preventing Zika virus infection in a human subject population in need thereof, comprising administering to individual human subjects of said human subject population a vaccine or immunogenic composition comprising antigen from a Zika virus, wherein the vaccine or immunogenic composition is administered as single dose or prime administration and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the single dose or prime administration a seroconversion rate of at least 25%, in a population of at least 20 Zika virus seronegative human subjects, as determined by the plaque reduction neutralization test (PRNT).
 71. The method of claim 70 wherein within 14 and/or 28 days after the single dose or prime administration a seropositivity rate of at least 25%, is induced in a population of at least 20 Zika virus seronegative human subjects or in a population of at least 20 Flavivirus naïve human subjects, as determined by the plaque reduction neutralization test (PRNT).
 72. The Method of claim 70, wherein the vaccine or immunogenic composition is administered as a single dose administration or multi dose administration including at least a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the administration induces systemic side effects in less than 50% of a human subject population of at least 20 flavivirus naïve human subjects or in a population of at least 20 Zika virus seronegative human subjects.
 73. The method of claim 69, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart, and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the boost administration geometric mean neutralizing antibody titers in a population of at least 20 flavivirus naïve human subjects and/or in a population of at least 20 Zika virus seronegative human subjects of greater than 300, as determined by the plaque reduction neutralization test (PRNT).
 74. The method of claim 70, wherein the vaccine or immunogenic composition is administered as a first (prime) and a second (boost) administration about 1 to about 16 weeks apart and wherein the administration of the vaccine or immunogenic composition induces 14 and/or 28 days after the boost administration a seroconversion rate of at least 70% in a population of at least 20 Zika virus seronegative human subjects, as determined by the plaque reduction neutralization test (PRNT), and a seropositivity rate of at least 70% in a population of at least 20 Zika virus seronegative human subjects or in a population of at least 20 Flavivirus naïve human subjects, as determined by the plaque reduction neutralization test (PRNT).
 75. The method of claim 70, wherein the administration of the vaccine or immunogenic composition until 7 days after the administration induces headache symptoms in less than 29% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 76. The method of claim 70, wherein the administration of the vaccine or immunogenic composition until 7 days after the administration induces fever in 4% or less, and/or fatigue in 33% or less, and/or arthralgia in 10% or less, and/or myalgia in 17% or less, and/or malaise in 15% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 77. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces systemic side effects in less than 40% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 78. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces at least 40% less, or at least 45% less fatigue, and/or no more fever, and/or no more, or at least 10% less, or at least 20% less, or at least 25% less myalgia, and/or no more, or at least 10% less, or at least 20% less malaise compared to 7 days after the prime administration in a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 79. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fever in less than 4% or in 0% of a human subject population of at least 20 flavivirus naïve human subjects or at least 20 Zika virus seronegative human subjects.
 80. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces fatigue in less than 19% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 81. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces myalgia in less than 12% or less than 8% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 82. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces malaise in less than 13% or in 10% or less of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 83. The method of claim 70, wherein the vaccine or immunogenic composition is administered as multiple doses in a first (prime) and a second (boost) administration and wherein the administration of the vaccine or immunogenic composition until 7 days after the boost administration induces headache symptoms in 20% or less, and arthralgia in 8% or less, and fever in less than 4%, and fatigue in less than 19%, and myalgia in less than 12%, and malaise in less than 13% of a human subject population of at least 20 flavivirus naïve human subjects or of at least 20 Zika virus seronegative human subjects.
 84. The method of claim 70, wherein the vaccine or immunogenic composition comprises a dose of 1 μg to 40 μg of the antigen, wherein the antigen is an inactivated whole virus.
 85. The method of claim 84, the Zika virus having a mutation at position 98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ ID NO:
 1. 86. The method of claim 70, wherein the antigen is purified, and wherein the main peak of the purified antigen in the size exclusion chromatography is more than 85% of the total area under the curve in the size exclusion chromatography.
 87. The method of claim 70, wherein the vaccine or immunogenic composition is administered to human subjects from a Zika endemic region, optionally subject to an outbreak.
 88. The method of claim 70, wherein the vaccine or immunogenic composition is administered to human subjects from a Zika non-endemic region travelling to an endemic region.
 89. The method of claim 70, wherein the vaccine or immunogenic composition is administered to human subjects 18 to 29 years of age, in particular women of childbearing potential.
 90. The method of claim 70, wherein the vaccine or immunogenic composition is administered to human subjects 30 to 49 years of age, in particular women of childbearing potential. 